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Western Blotting:
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Blotting: Introductions
2 Western blotting: Surveying the past to the present
A Guide Tianna Hicklin, Ph.D.
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Methods
3 A new standard in reproducible quantitative
Western blotting
Kim Cushing
Product Marketing Manager
GE Healthcare

Western blotting overview


4 A history of the old West(ern blot)

7 Preparing for your Western blot

10 Choosing your detection method

Since
Research Articles
1880 12 An analysis of critical factors for quantitative
immunoblotting
Kevin A. Janes

22 A neuronal antigen in the brains of Alzheimer patients


Benjamin L. Wolozin, Alex Pruchnicki, Dennis W. Dickson, et. al

25 Serological analysis of a subgroup of human


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W E S T E RN BLOT T ING : A G U I D E TO CU R R E N T M E TH O D S S E C T I O N O N E | I NT R O D U C T I O NS

W W
estern blotting—easily recognizable by the telltale estern blotting, also known as immunoblotting when
images of laddered bands amongst the pages of first described in 1979, is today a ubiquitous method
scientific journals—has remained a relatively popular in the life science laboratory. From cell biology to
protein detection technique over the past 35 years. protein purification and characterization, Western
From immunology to neuroscience to drug discovery, the fields to blotting remains an essential protein analytical technique that is fun-
which Western blots have contributed are wide ranging. Science and damental to protein research.
its family of journals have published hundreds of research articles that Although the details may vary from application to application,
have used the technique since it was first described, as it has been Western blotting protocols all follow some common steps: sample
a useful tool in many scientific discoveries. Shortly after the method preparation, gel electrophoresis to separate the proteins, transferring

Western
was first described in 1979, for example, Western blots began being
used to unravel questions about which proteins were contributing to
A new the proteins to a membrane, blocking of the membrane, targeting the
protein of interest with a specific primary antibody, detection using a

blotting:
drug resistance (see page 28) and Alzheimer’s disease (see page 22)
as well as to characterize the human retrovirus that was causing a new standard in labelled secondary antibody, and finally imaging and analyzing the
results.

Surveying
epidemic, AIDS (see page 25).
Though it has remained a staple for protein detection over the reproducible One of the biggest challenges for Western blotting is data
reproducibility. Within the multistep immunoblotting process, there
are many potential ways to introduce error and variation. This inherent
the past to
years, the technique is not immune from the growing concerns of
the scientific community for increased reproducibility of data. Such quantitative risk often necessitates that experiments are repeated many times in
order to generate reproducible data. Every step of the process has
the present
concerns have been recently described in an editorial by Science
Journals Editor-in-Chief Marcia McNutt in which she discussed the Western an impact on the result and must be controlled to ensure the quality
of the final data. This is particularly critical when data publication is
importance of reproducibility, and transparency, when publishing
data (www.sciencemag.org/content/346/6210/679.full) as well as the blotting the ultimate goal and reproducible quantitative data is an absolute
prerequisite.
efforts a group of editors put forth to design guidelines for reporting
data in the hopes that they will be viewed “as part of the quality con- In 1990, Amersham International (now part of GE Healthcare) was
trol that justifies the public trust in science.” the first company to introduce enhanced chemiluminescent (ECL)
For Western blots in particular, the optimization of various steps Western blotting detection reagents. Chemiluminescence is a highly
and a great deal of experience working with the technique are key to sensitive detection methodology and was the leading detection
gathering quality data that is reliable and reproducible. As a multistep technology used in Western blotting for many years; however, as the
process, the technique inherently houses many places in which vari- critical horseradish peroxidase enzyme reaction is dynamic, the light
ability can be introduced during data collection. In a 2015 Science produced declines over time making it challenging to produce truly
Signaling article, Kevin A. Janes details some of the critical factors for quantitative data.
achieving accurate immunoblotting data, including sample prepara- Advances in charge-coupled device (CCD) technology have
tion, loading controls, and choice of reagents and buffers (see page led to the use of digital imaging systems that can capture both
12). chemiluminescent and fluorescent signals. When using fluorescence
In this booklet, we invite you to explore some of the history of detection, the signal generated by dye excitation is stable for several
Western blotting as we take a look back at some of the highly cited months, producing consistent and quantitative results, making it an
advances to which the method has contributed and explore some of excellent choice for true protein quantitation.
the many ways the technique can be optimized to help create higher Since the introduction of the first ECL detection reagent for
quality and more reproducible data. Western blotting, the portfolio of products offered by GE Healthcare
has been expanded, improved, and optimized to cover the breadth of
Tianna Hicklin, Ph.D. Western blotting requirements from sensitive protein identification to
Editor, Science/AAAS Custom Publishing Office reproducible protein quantification. The most recent addition to the
range is the new Amersham WB system, which integrates separation,
transfer, detection, and quantitative analysis of proteins. This fully
integrated system minimizes assay variability to provide consistent,
quantitative Western blot data for every sample, every time.
Western blotting has played a central role in many applications
over the last 36 years, which this guide illustrates. It continues to
be an essential technique which, while undergoing significant
improvements remains largely faithful to the tenet of the original
protocol. There are opportunities to optimize and refine the Western
blotting protocol that can deliver some real benefits and this Western
blotting guide points to some of the key considerations which can
help in this regard. We are delighted to sponsor this publication.

Kim Cushing
Product Marketing Manager
GE Healthcare

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W E S T E RN BLOT T ING : A G U I D E TO CU R R E N T M E TH O D S S E C T I O N T W O | WESTERN BLOTTING OVERVIEW

A history of the old


gel, are transferred to a membrane such that their pattern in the Ling’s team didn’t To find out, Robert
gel is retained, like making a photocopy, or what Towbin called use that antiserum Gallo and his team at
“a faithful replica” (1). That “blot” is then probed using “primary” directly, however; the National Cancer

West(ern blot) antibodies to a specific protein or proteins of interest, and de-


veloped by addition of secondary antibodies, which recognize
they cleaned it up first
by preabsorbing it
Institute in Bethesda,
Maryland, performed
the primaries. These secondary antibodies are labeled with against a preparation a battery of tests,
By Jeffry M. Perkel either a radioisotope, fluorophore, or enzyme, all of which allow from drug-sensitive including electron
the proteins’ positions to be identified. cells. In this way, microscopy, reverse
The method is largely unchanged—at least in broad strokes— they were able to transcriptase enzyme
since Towbin, with Theophil Staehelin and Julian Gordon, used clearly detect a band assays, cell co-culture
it to detect bacterial or chicken ribosomal proteins that had consistent with the experiments—and
been separated on urea-containing polyacrylamide gels. They P-glycoprotein in drug- Western blots. In one

W
hen Harry Towbin and colleagues at the Fried- detailed their method in the Proceedings of the National Acad- resistant but not sensi- series of experiments,
rich Miescher Institute in Basel, Switzerland first emy of Sciences in 1979, proposing such applications as screen- tive lines. the team infected
detailed the procedure that would come to be ing for monoclonal antibody-producing hybridomas, autoim- But those results, human T cells with
called the Western blot, almost nothing was easy. mune sera, enzymatic activities, and ligand binding (1). the researchers ac- the virus that they
Antibodies weren’t readily available in 1979, so they made their Yet Towbin’s original article never actually names the meth- knowledged, were still called HTLV-III, then
own by injecting purified ribosomal proteins into mice or goats. od; it simply refers to an “electrophoretic blotting technique.” only correlative – they separated cellular
Their transfer procedure involved a gel and nitrocellulose mem- The term “Western blot” was actually coined 2 years later by had no way of know- proteins (including the
brane sandwiched between two Scotch-Brite scouring pads W. Neal Burnette, of the Fred Hutchinson Cancer Research ing, for instance, whether the P-glycoprotein gene was trans- virus) on protein gels and blotted them to nitrocellulose. They
and a “disposable micropipette tray” for structural support, and Center in Seattle, Washington, who described several technical ferred with other sequences that actually conferred the drug then cut those blots into strips and “tested [them] with samples
bound together with rubber bands. The transfer itself was con- improvements to the method, including applying it to the more resistance itself. Much of that doubt, though, was eliminated of human serum in a strip radioimmunoassay (RIA) based on the
ducted in “an electrophoretic destaining chamber” (1). commonly used SDS-PAGE gels: when the researchers demonstrated in 1983, again via Western Western blot technique” (5; see page XX). Those sera samples
Secondary antibodies were a little easier, at least for fluores- blotting, that drug resistant cells from Chinese hamster, Syrian had been taken “from patients with AIDS or pre-AIDS, from
cent and colorimetric detection since these could be purchased. “With due respect to [Edwin] Southern, the established hamster, mouse, and human all contained the 170 kD P-glyco- contacts of such patients, and from homo- or heterosexual male
But for radioisotopic detection, the researchers once again were tradition of ‘geographic’ naming of transfer techniques protein, all of which could be detected using antibodies raised controls.”
on their own, labeling their own antibody preparations with (‘Southern,’ ‘Northern’) is continued; the method against membrane preparations from both drug-resistant CHO The goal was to determine whether blood samples collected
iodine-125 via “the chloramine T method”—and exposing the la- described in this manuscript is referred to as ‘Western’ and human cell lines (4; see page XX). from patients with AIDS contained antibodies capable of
beled blots to film for 6 days. blotting” (2). These data, of course, suggested that whatever the recognizing HTLV-III proteins, and as it turns out, they did.
Today, the method is considerably easier: researchers can P-glycoprotein is, it is evolutionarily conserved. But the Sera from patients with AIDS or pre-AIDS lit up specific bands
purchase precast gels, transfer proteins to membranes using Unraveling multidrug resistance implications were broader than that. “Our findings could in virus-infected cellular lysates, but not uninfected control
commercial transfer apparatuses, and capture their data in One of the earliest applications of Towbin’s procedure used have important implications for cancer therapy,” Ling wrote in cell lines. Control patient sera did not bind HTLV-III proteins.
seconds using digital imagers. Towbin’s landmark paper has the method to tease apart the mechanism of multidrug resis- Science. “It is possible that clinical resistance to combination The analysis also demonstrated that HTLV-III is distinct from,
been cited more than 51,000 times in the intervening 36 years, tance in immortalized cells. chemotherapy might result from an unchecked proliferation though related to, other HTLV family members, and began
according to Google Scholar. Yet for the most part, the method Researchers had observed that while many cultured cells of tumor cell subpopulations with a multidrug resistance the painstaking process of teasing apart the virus’ molecular
remains largely the same. Modern Western blot sandwich were sensitive to drugs like colchicine and actinomycin D, some phenotype” (4). composition (5).
cassettes bear a strong family resemblance to the one Towbin were resistant, and if they were resistant to one compound, they Today, that prediction has been shown to be true. The 170 In total, the Gallo team published four studies in the 4 May
dreamed up. And researchers still use fluorophore and enzyme- often were resistant to many. Molecular analysis determined kD P-glycoprotein is an ATP-binding cassette (ABC) transporter 1984 issue of Science. In the final one, the team determined that
conjugated secondary antibodies to identify bands of interest, that a 170 kD membrane protein seemed to correlate with mul- that actively pumps drugs and other compounds out of blood sera from 43 of 49 AIDS patients and 11 of 14 pre-AIDS
though most researchers now favor chemiluminescence over tidrug resistance, but it was not yet clear if that protein was ac- cells, conferring drug resistance to cancer cells—a clinically patients recognize antigens—particularly the 60 kDa env protein—
colorimetric detection. tually causative of the phenotype, or merely a passenger. important observation that was made possible via the Western found in a viral extract from HTLV-III-producing cells, in this case
That’s not to say methods developers have been content to In 1982, Victor Ling of the Ontario Cancer Institute and blot. using ELISA and RIA based on the Western protocol (6).
rest on their laurels. Researchers have extended and evolved University of Toronto, and colleagues demonstrated that DNA “The data presented here and in the accompanying reports
the method over the decades, making it easier, faster, and more transfer from drug-resistant cells to sensitive cells induced Decoding AIDS suggest that HTLV-III is the primary cause of AIDS,” Gallo and
reliable. a resistant phenotype, including the appearance of that 170 Another landmark in Western history occurred in 1984. At his team concluded (6). Within a few years, HTLV-III would be
Here, we travel back to the Old Western, to see where the ven- kD protein on polyacrylamide gel separations of membrane the time, acquired immunodeficiency syndrome (AIDS) was rebranded with the name by which it is known today: human im-
erable immunoblot has been and explore how far the technique protein preparations. But the band was diffuse and partially a disease of unknown etiology that was impacting mostly munodeficiency virus (HIV).
has come. obscured by the complex membrane material, so, “To increase intravenous drug users and homosexual men. The disease had That discovery, wrote Jean Marx in a news story accompany-
the sensitivity and specificity of detecting the P-glycoprotein,” only been recognized in 1981, but within 2 years, researchers ing the reports, opened the door to the development of re-
The Western blot they wrote, “we employed an antiserum raised against CHO had determined that the causative agent likely was a retrovirus, agents for disease diagnostics and blood donor testing, as well
A Western blot is simply a way to identify proteins on a poly- [Chinese hamster ovary] cell mutant membranes in a replica a relative of the human T-lymphotropic viruses (HTLV). The as for research into potential vaccines (7). And Western blotting
acrylamide gel. Proteins, separated by size on a polyacrylamide Western blot procedure” (3). question was: which one? played a key role. continued>

4 sciencemag.org SCIENCE SCIENCE sciencemag.org 5


W E S T E RN BLOT T ING : A G U I D E TO CU
M ORDREERNNTM
MEETH
THOODDSS S E C T I O N T W O | WESTERN N O N E | EDITORIAL
S E C T I OBLOTTING OVERVIEW

Preparing for your


Untangling Alzheimer’s disease so-called “synaptosome,” a structure involved in synaptic vesicle with some 1.8 million antibodies listed in Antibodypedia, it’s a
In 1986, researchers used Western blotting to address yet recycling. They then combined those figures with electron mi- good bet somebody else has already done so. Meanwhile, us-
another clinical puzzle, Alzheimer’s disease. croscopy, mass spectrometry, and superresolution imaging data ing digital gel-documentation systems researchers can ditch
In an effort to begin to understand the biochemistry of dis-
ease, Benjamin Wolozin and colleagues at the Albert Einstein
to generate “a three-dimensional model of an ‘average’ synapse,
displaying 300,000 proteins in atomic detail” (12).
their manual cameras and lightrooms and capture digital data
instead. Such systems certainly are easier to use than X-ray film, Western blot
College of Medicine used brain extracts from control patients As its applications have evolved, so too has the Western blot- the traditional method of chemiluminescent detection, but they
and patients with Alzheimer’s to identify an antibody that could ting method itself. Shortly after the original Towbin paper, for also represent a tremendous advance in that they are more By Jeffrey M. Perkel
distinguish diseased and non-diseased brain homogenates. instance, researchers developed the so-called Southwestern sensitive, produce a linear signal over a wider dynamic range,
The resulting reagent, called “Alz-50” bound an antigen that blot (13). Literally a cross between Southern and Western blot- and enable more reproducible experiments. Equally significant
was some 15 to 30 times more abundant in the temporal cortex, ting, the technique probes protein blots with labeled DNA to is the development of methods to make what effectively is a
hippocampus, and nucleus basalis of patients with Alzheimer’s identify DNA-binding proteins. qualitative technique, quantitative—a development that pow-
disease than non-disease individuals (8; see page XX). Alz-50 Another variation is the “In-Cell Western,” combining the

W
ered the synaptosome modeling study, for instance (12, 14; see
features of Western blotting and ELISA. In a traditional ELISA, page XX). estern blotting is ubiquitous in modern molecu-
an antibody to the target of interest is bound to a solid surface, The remarkable thing is, for all these advances, the original lar biology laboratories. It’s relatively simple to
mixed with a sample, and then probed with a second, labeled Towbin protocol remains apparent. Countless labs around the perform, but good-looking data—and especially
Researchers have extended and evolved antibody. In an In-Cell Western, cells grown on the surfaces of world still use plastic Western blotting cassettes and sponges quantitative and reproducible data—can be hard to
microtiter plates are lysed in situ, probed with primary antibod- that look remarkably like the sketch in the seminal 1979 paper. come by. A complete Western blotting protocol comprises doz-
the method over the decades, making it ies to the antigen(s) of interest, and finally detected using fluo- The process is more efficient and straightforward than ever, ens of steps, dozens of places in which something can go wrong,
easier, faster, and more reliable. rescently labeled secondary antibodies. The method avoids the yet the fundamental principle of creating and probing a from sample preparation all the way through to detection. Mak-
need for gel electrophoresis and blotting, and thus is faster than “faithful replica” of a protein gel remains. As the Western blot ing a protocol that is reproducible from day to day and lab to lab
a traditional Western. But it also assumes no background bind- closes in on four decades, it will be interesting to see where it such that researchers can be confident of anything but the most
stained neurons in the brains of patients with Alzheimer’s but ing, as there is no electrophoretic separation step. goes next. dramatic differences in abundance can be a challenge. Here we
not control individuals, and also lit up the neurofibrillary tangles Methods developers have also found ways to accelerate and review some key steps to consider.
that characterize the disease. optimize the Western procedure. As detailed in a Science maga- Jeffrey M. Perkel is a freelance science writer based in Pocatello,
The question was, to what antigen was Alz-50 binding. zine technology feature, for instance, EMD Millipore’s SNAP i.d. Idaho. Sample preparation
Wolozin and colleagues enriched the antigen from Alzheimer 2.0 system uses a vacuum manifold to reduce Western blotting The first step in a Western blotting protocol is preparing a
and control brain homogenates, resolved them on an SDS- incubation and washing steps “from 4 hours to 30 minutes” (9). References cell or tissue lysate. The cell membranes must be broken open
polyacryl amide gel, transferred to nitrocellulose, and probed Others have developed methods to reduce blotting time and 1. H. Towbin et al., Proc. Natl. Acad. Sci. U.S.A. 76, 4350 to release the cellular contents, using a lysis or extraction buffer,
the blot with Alz-50. Detection, using an alkaline phosphatase- increase throughput, such as ProteinSimple’s Simple Western (1979). and the protein(s) of interest solubilized so they can be sepa-
conjugated secondary antibody, revealed a 68 kD pro tein in system. Simple Westerns replace the traditional SDS-PAGE 2. W. N. Burnette, Anal Biochem. 112, 195 (1981). rated with gel electrophoresis. But there is no one best way to do
diseased brain homogenates that was absent in normal tissue gel and blotting steps with capillary electrophoresis, which 3. P. G. Debenham et al., Mol. Cell Biol. 2, 881 (1982). that. Some samples, for instance, require harsher lysis conditions
(8). can separate proteins by size or charge. Up to 96 samples can 4. N. Kartner et al., Science 221, 1285 (1983). than others, and not all proteins are soluble or stable in the same
“Our results demonstrate that Alz-50 recognizes a protein automatically be run in parallel and detected in the capillaries 5. J. Schüpbach et al., Science 224, 503 (1984). detergent.
present in neuronal terminals in plaques and in most neurons directly via chemiluminescent detection. The resulting data are 6. M. G. Sarngadharan et al., Science 224, 506 (1984). Lysis buffers “differ in their ability to solubilize proteins, with
with tangles,” Wolozin et al. concluded. But they had no idea then rendered as a traditional blot using the system software. 7. J. L. Marx, Science 224, 475 (1984). those containing sodium dodecyl sulfate [SDS] and other ionic
what that protein might be, though they doubted it could Another Western blotting optimization that has emerged 8. B. L. Wolozin et al., Science 232, 648 (1986). detergents considered to be the harshest and therefore most
be tau protein, one of the few proteins known to be of the since Towbin is multiplexing. Researchers using fluorophore- 9. A. Harding, Science, (2013), doi:10.1126/science.opms. likely to give the highest yield,” according to a guide published
right size, as it did not exhibit the differential abundance they conjugated secondary antibodies now can detect two or more p1300073. by antibody-developer Abcam (1). Some common nonionic
observed. Today, researchers know that presumption was in- antigens simultaneously, which is particularly useful for quanti- 10. V. J. Starai et al., Science 298, 2390 (2002). detergents include Triton X-100, Nonidet P-40, and Tween; ionic
correct: Alz-50 binds a phosphorylated form of tau, a major tation and normalization of protein abundance (see Preparing 11. G. Legname et al., Science 305, 673 (2004). detergents include SDS, deoxycholate, and hexadecyltrimethyl-
component of neurofibrillary tangles in so-called tauopathies, for your Western blot, page XX). In one typical example, the 12. B. G. Wilhelm et al., Science 344, 1023 (2014). ammonium bromide (CTAB).
including Alzheimer’s. antigen of interest is detected using one fluorescent color and a 13. F. K. Y. Siu et al., Nat. Protocols 3, 51 (2008). In a recent report in Science Signaling, Kevin Janes of the Uni-
housekeeping protein with a second. Because both proteins are 14. K. A. Janes, Science Signaling 8, rs2 (2015). versity of Virginia documented several “critical factors for quan-
Technical innovations detected on a single gel and simultaneously, the method saves titative immunoblotting,” including sample preparation (2; see
Western blotting has since participated in studies across time. But it also improves reproducibility relative to the alterna- page XX). He noted, for instance, that the commonly used RIPA
the biological spectrum, from identifying the substrates of the tives, which are either to probe, strip, and reprobe the blot with buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100,
aging-associated Sir2 deacetylase and probing the biology of a second primary antibody, or to run duplicate gels and probe 0.5% sodium deoxycholate, 0.1% SDS, 5 mM EDTA, plus protein-
synthetic prions to modeling neurotransmitter synaptic vesicle them with separate antibodies. ase and phosphatase inhibitors)—efficiently solubilizes cellular
trafficking (10–12). Perhaps the most significant technical advances in Western proteins, though some are left behind in an insoluble pellet.
In the latter study, Silvio Rizzoli of the University of Göttingen blotting history, however, have been the most pedestrian. For In Janes’ own hands, analysis of the soluble and insoluble
Medical Center, and colleagues, used “quantitative immunob- one thing, unless they are studying a new protein, researchers fractions resulting from RIPA lysis of HT-29 human colon
lotting” to measure the abundance of each of 62 proteins in the rarely need, as Towbin did, to prepare their own antibodies; adenocarcinoma cells revealed that though continued>

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W E S T E RN BLOT T ING : A G U I D E TO CU R R E N T M E TH O D S S E C T I O N T W O | WESTERN BLOTTING OVERVIEW

“many cytoplasmic proteins” were efficiently solubilized in interest to a control, such as a housekeeping protein. The latter tends to (TBST or PBST) (6),
RIPA, some histones and transcription factors were confined But that assumes the housekeeping protein’s abundance be sturdier and have but the number,
to the insoluble fraction. Cleaved caspase 8, an apoptotic is constant across conditions— an assumption that must be a higher protein- duration, and
indicator, was soluble in Laemmli sample buffer (62.5 mM tested. Also, researchers must test to ensure that both the binding capacity, but stringency of washes
Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 100 mM dithiothreitol, housekeeping protein and protein of interest produce linear, may also yield higher can vary, as can the
0.01% bromophenol blue), but not in RIPA or NP-40 buffer (50 proportional responses, such that a two-fold change in protein auto fluorescence amount of detergent
mM Tris-HCl pH 8.0, 150 mM NaCl, 0.5% NP-40, 5 mM EDTA, abundance yields a twofold change in signal. depending on used. Antibody
plus inhibitors). Other options include Tris-Triton buffer for In one recent example, Alicia McDonough and colleagues membrane type. dilution also is key—
cytoskeletal proteins (10 mM Tris pH 7.4, 100 mM NaCl, 1 mM at the Keck School of Medicine at the University of Southern On the other hand, you need sufficient
EDTA, 1 mM EGTA, 1% Triton X-100, 10% glycerol, 0.1% SDS, California, loaded a gel with kidney homogenate samples. nitrocellulose is antibody to detect
0.5% deoxycholate) and 20 mM Tris-HCl pH 7.5 for soluble Some lanes contained 3 mg of the lysate; others contained more compatible the target, but not
cytoplasmic proteins (1). twice as much. The team then probed with antibodies for with most protein so much as to create
claudin-10 and actin. The claudin signal was twice as intense stains that enable high background on
in the high-abundance lanes, indicating that the protein was in visual inspection of the blot, which can
a linear detection range. But actin, a common control protein, transfer efficiency. obscure faint bands.
Researchers can cast their own gels produced an equivalent signal in both cases, indicating that it Once your pro- The secondary
“is not an acceptable loading control for this application” (3). teins are transferred antibody is likewise
for separating proteins, but they One possible solution is normalizing against multiple to a membrane, critical, so be
also can buy them precast. That offers controls. For instance, Janes showed in his study that the blocking reagents sure to optimize
apparent abundance of phosphorylated Smad2 protein are used to reduce your dilution
a speed advantage, but it’s also changes dramatically depending on the choice and number antibodies nonspe- and incubation
of normalization controls he used (he tested as many as five at cific binding on the conditions there, as
safer and more reproducible. once). “As higher-order combinations of loading controls were membrane. Com- well.
tested as normalizers, I found that the coefficient of variation of mon options include Like all methods,
In a separate experiment, Janes illustrated the importance of p-Smad2 linker steadily improved toward 7% to 8%,” he wrote nonfat milk and bovine serum albumin, but several commercial Western blotting has both strengths and weaknesses. The
phosphatase inhibitors during lysis in probing various members (2). formulations exist too. In many cases, any blocker will do, but it technique is easy enough that almost anyone can perform it.
of the “Akt-glycogen synthase kinase-3 (GSK3)-glycogen An alternative approach normalizes each band’s intensity can be antibody dependent. Milk is suboptimal for phosphoryla- The question is: how well does that signal reflect the cells it
synthase (GS) signaling axis.” In particular, he showed that to total protein in the lane using either a general protein stain, tion analysis, for instance, because milk contains casein, a phos- represents? With careful experimental design and rigorous
some post-translational modifications were more stable in the such as Ponceau S, Memcode, or fluorescent dye. McDonough phoprotein that can compete with the target for antibody bind- control procedures, researchers can be sure it’s as close as
absence of inhibitors in NP-40 lysis buffer than RIPA. recommends running a parallel “loading gel, which is loaded ing and reduce specific signal intensity. Milk also may contain possible.
“These results collectively showed that lysis buffer and run identically to the gel(s) that will be immunoblotted” (3). biotin and glycoproteins that will light up with streptavidin-based
composition substantially affects the results of quantitative The gel is stained and imaged, and the intensities of several detection schemes (4, 5). When in doubt, try several options to Jeffrey M. Perkel is a freelance science writer based in Pocatello,
immunoblotting,” Janes wrote (2). discrete bands averaged for normalization. Alternatively, see which works best for your system. Idaho.
the samples can be prelabeled prior to electrophoresis
Normalization and the total signal on the membrane after transfer can Antibodies References
Western blotting typically is used to measure changes be used as loading control. This is especially suitable for The key reagent in a Western blot, of course, is the primary 1. Abcam, “Sample preparation (WB guide),” [http://www.
in protein abundance across different conditions. For such fluorescent Western, since the target signals can be detected antibody used to recognize the protein of interest. Thousands abcam.com/index.html?pageconfig=resource&rid=11379
comparisons to be meaningful, each lane should contain the simultaneously on the membrane by multiplexing. of antibodies are commercially available, but not all are #A1]
same amount of protein, ideally representing equivalent cell equally good. Look for antibodies that have been validated as 2. K.A. Janes, Science Signaling 8:rs2 (2015).
numbers. Electrophoresis and blotting compatible with Western blotting—typically, these recognize 3. A.A. McDonough et al., Am. J. Physiol. Cell Physiol. 308,
First, researchers need to know how much protein they have Researchers can cast their own gels for separating proteins, linear peptides as opposed to conformational epitopes, as C426 (2015).
in each sample. The Abcam guide recommends quantifying but they also can buy them precast. That offers a speed most Westerns are run under denaturing conditions. (Native 4. LI-COR Biosciences, “Optimizing chemiluminescent
protein concentration using “a Bradford assay, a Lowry assay advantage, but it’s also safer—unpolymerized polyacrylamide is Westerns, of course, retain protein conformation). You may need Western blots,” 2011. [biosupport.licor.com/docs/Chemi_
or a BCA [bicinchoninic acid] assay,” usually with bovine serum neurotoxic—and more reproducible. Precast gradient gels can to test several primaries to see which works best, but be sure Good_Westerns.pdf]
albumin (BSA) as a protein standard (1). be especially useful, providing particularly sharp resolution over also to demonstrate that the antibody actually is specific for 5. GE Healthcare Life Sciences, “Western Blotting Principles
Then, because minor pipetting errors during loading the specific size ranges; such gels are challenging to create from your antigen in the first place—that is, that is produces a band of and Methods,” 2011. [www.gelifesciences.com/file_source/
gel or errors when measuring protein concentration can scratch in a reproducible fashion. Precast gels also are available the correct size in positive control samples and not in negative GELS/Service%20and%20Support/Documents%20and%20
dramatically over- or under-emphasize biological effects, for analysis of nondenatured (i.e., “native”) proteins. These are controls (3). Downloads/Handbooks/pdfs/Western%20Blotting.pdf]
researchers should also take steps to ensure an equal amount useful especially when studying intact protein complexes or Then, optimize how much and how long blots are incubated 6. Cell Signaling Technology, “Western blotting
of protein is present in each lane. using antibodies that recognize conformational epitopes. with an antibody and the washing conditions used. Typical protocol,” [http://www.cellsignal.com/common/content/
In one common approach to minimize the effect of loading Western blots typically use either nitrocellulose or wash conditions are three washes of 5 minutes each in Tris- or content.jsp?id=western].
variation, researchers compare the intensity of their protein of polyvinylidene fluoride (PVDF) membranes for protein blotting. phosphate-buffered saline (TBS or PBS) containing Tween 20

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W E S T E RN BLOT T ING : A G U I D E TO CU R R E N T M E TH O D S S E C T I O N T W O | WESTERN BLOTTING OVERVIEW

Choosing your
from now, assuming the blot is properly handled and stored. ping can also remove target colleagues described a quanti-
Chemiluminescent signals, in contrast, are transient and variable. proteins. Alternatively, re- tative assay for the detection of
Researchers performing fluorescence-based Westerns also searchers can run and probe Plasmodium falciparum circum-

detection method have the ability to combine two or more secondary antibodies
to detect multiple proteins at once, a process called multiplex-
replicate blots in parallel, but
this, too, is problematic, as
sporozoite protein (PfCSP), a
potential vaccine antigen.
ing. Multiplexing—which requires only that the different primary the blots may not be treated The assay, the authors report,
By Jeffrey M. Perkel antibodies be raised in different organisms and that the corre- identically. was linear between about 3 and
sponding secondaries be conjugated to spectrally distinct dyes— Complicating quantifica- 12 pg of protein, making it “the
reduces an experiment’s hands-on time and improves quantita- tion of chemiluminescent most sensitive assay for immu-
tive accuracy and reproducibility relative to chemiluminescence, Westerns is the detection noassay-based detection of
which is strictly a singleplex technique. mechanism itself. Chemilumi- PfCSP or any malarial protein.”
nescence stems from an enzy- (ELISA assays, by comparison,
matic reaction that consumes

I
Quantitative vs. qualitative detection required at least 100 ng of pro-
n their seminal Western blotting paper, Towbin et al. devel- By default, Western blotting is inherently qualitative—a protein a finite supply of substrate. tein for detection.) Furthermore,
oped their blots using radioisotopic labeling, colorimetric is either present, or it is not. But researchers frequently use the Thus, the signal intensity of the authors found the assay to
detection, or fluorescence (1). Thirty-six years later, the technique to assess quantitative differences, such as how protein a chemiluminescent blot will be highly reproducible, with
method has changed remarkably little. But one thing that has abundance changes under different conditions. vary with time and substrate concentration. A 30 second expo- an inter-assay coefficient of variation (CV) of 10.31% and a mean
changed is how the experiment is read out: Modern researchers Fundamentally, converting a band’s intensity into a reliable sure taken immediately after substrate addition will look very dif- intra-assay CV of 3.16% (3). But to achieve that level of reproduc-
most commonly develop their Western blots using chemilumi- and meaningful assessment of abundance requires that the over- ferent from an exposure of the same duration collected 10 min- ibility, the authors had to go beyond the typical Western protocol,
nescence detection. all amount of the protein does not vary from lane to lane, that the utes later. It may also vary if the substrate reagent is a little older, using serial dilutions of protein preparations, standard curves,
Fluorescence-based detection—which in Towbin’s study was signal any band generated be linearly related to that protein’s or doesn’t cover the blot uniformly. As a result, it can be difficult and multiple replicates.
the least sensitive method tried—has also evolved into an increas- abundance, and that the detector’s ability to capture that signal to acquire an “ideal” exposure, as it isn’t necessarily obvious how
ingly sensitive and popular alternative. also is linear. Film, for instance, is a poor choice for quantitative different exposures will appear. Equipment and reagents
Both chemiluminescent and fluorescent detection offer sub- Western blotting, writes Kevin Janes of the University of Virginia, The situation can be particularly tricky in cases involving mul- Other differences between chemiluminescence- and fluo-
stantial advantages over the previously preferred options of colo- because its dynamic range “is so small that quantitative analysis tiple bands of dramatically different intensities. At the very least, rescence-based Westerns include the equipment and reagents
rimetry and radiolabeling, being more sensitive than the former is virtually impossible. Film can make small differences in abun- researchers may end up wasting considerable time fine-tuning required to run the experiments. Nitrocellulose membranes, for
and less hazardous and troublesome than the latter. But they also dance appear as large differences in band intensity. When satu- their exposures to capture both bands in the detector’s linear instance, are compatible with both approaches, but are relatively
differ from each other in several important ways. rated, film exposures can also hide sample-to-sample variation range, such that their intensities accurately reflect their abun- fragile. Polyvinylidene fluoride (PVDF) membranes are sturdier,
for high-abundance proteins such as loading controls” (2; see dance. Experimental timing becomes key in such situations, and but exhibit lower sensitivity and higher autofluorescence. Dedi-
Chemiluminescence vs. fluorescence page XX). replicating such conditions from day to day requires technical cated “low-fluorescence” membranes are commercially available
In chemiluminescence, the secondary antibody—the antibody One way to ensure band intensity accurately reflects protein skill. Even then, it isn’t easy to compare one “ideal” image with to address these shortcomings.
that allows researchers to detect the antibody binding to the pro- abundance, and to account for sample-to-sample variation, is another captured from a different blot, on a different day, so re- Chemiluminescence can be detected in most standard gel
tein of interest—is conjugated to an enzyme (usually horseradish multiplexing. Rather than assessing protein abundance from searchers often are advised against direct comparisons between documentation systems, and even on X-ray film (though that is
peroxidase (HRP)), just as in Towbin’s original study (1). But where the intensity of a single band in isolation, researchers typically experiments. not advised, given film’s poor sensitivity and narrow dynamic
Towbin and colleagues detected their HRP-tagged antibodies via compare it to one or more invariant normalization controls, which In contrast, fluorescence signals are largely quantitative, so a range). Fluorescence, however, requires a dedicated instrument,
the deposition of a colored precipitate from a colorless substrate, usually are housekeeping proteins (see Preparing for your West- band with twice the intensity of another band can generally be such as a laser scanner or CCD camera equipped with suitable
chemiluminescence uses hydrogen peroxide to oxidize luminol, ern blot, page XX). assumed to contain twice as much material. light sources and emission filters.
producing a transient burst of photons that must be detected us- In a fluorescent Western, this is easily accomplished. A re- In his recent study on the factors influencing the reliability Given all these variables, which option should you choose?
ing autoradiography or a digital imager. searcher could use a goat anti-rabbit secondary coupled to Cy3 of quantitative Western blotting, Janes wrote that his lab favors In many cases, it really doesn’t matter. Researchers interested in
Fluorescence detection uses secondary antibodies labeled to detect their protein of interest, and a rabbit anti-mouse sec- fluorescence over chemiluminescence Westerns “whenever pos- a qualitative assessment of their samples can often use either
with fluorescent dyes, which also are detected on a digital im- ondary conjugated to Cy5 (i.e., a dye with distinct excitation and sible” (2). chemiluminescence or fluorescence detection, depending on
ager. An alternative approach, chemifluorescence couples HRP emission spectra) to detect, say, tubulin. Using the fluorescence “I compared the linearity of [fluorescent dye]-conjugated the antibodies and equipment they have on hand. But for a deep
activity to formation of a fluorescent product. intensity of those two signals, they then can determine the abun- secondary antibodies to horseradish peroxidase conjugates that dive into protein quantitation, they may want to give fluorescence
Chemiluminescent Westerns generally are considered more dance of their protein in their experimental condition relative to were incubated with an enhanced chemiluminescence (ECL) a try.
sensitive than their fluorescent counterparts, with detection down the control—a ratio that is more reliably comparable from experi- cocktail or a commercial substrate marketed for high-sensitivity
to the low femtogram level in some cases. But as a practical mat- ment to experiment than the single protein’s absolute intensity. applications,” Janes wrote. “Under the same immunoblotting con- Jeffrey M. Perkel is a freelance science writer based in Pocatello,
ter, fluorescence Western blots are simpler to perform, as there’s The problem is, chemiluminescence doesn’t offer multiple ditions, chemiluminescent exposures consistently yielded stron- Idaho.
no chemiluminescent substrate to add (which saves time and detection channels. The only way to quantify multiple proteins in ger band densities. However, the linear dynamic range was very
money) and the signal is generally stable over time (simplifying a chemiluminescent Western is to strip off the primary antibody limited, and signals often decreased at high protein inputs” (2). References
data collection and enhancing reproducibility). (using, for instance, 6M guanidine-HCl), and reprobe the blot with That’s not to say quantitative chemiluminescence is impos- 1. H. Towbin et al., Proc. Natl. Acad. Sci. U.S.A. 76, 4350 (1979).
All things being equal, an exposure taken of a fluorescently another primary antibody against the second protein of interest— sible. In one 2013 report, Sanjai Kumar of the U.S. Food and 2. K. A. Janes Science Signaling 8, rs2 (2015).
labeled blot today will be exactly the same as one taken a week a process that adds time and impacts reproducibility, since strip- Drug Administration’s Laboratory of Emerging Pathogens, and 3. S. Kumar et al., J. Immunol. Methods 390, 99 (2013).

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An analysis of critical factors for Laemmli sample buffer (7), and then immu-
noblotted for 20 different protein targets. As

quantitative immunoblotting
expected, RIPA lysis buffer efficiently solubi-
lized many cytoplasmic proteins [glyceralde-
hyde-3-phosphate dehydrogenase (GAPDH)
and heat shock protein 90 (Hsp90)] and sig-
Kevin A. Janes naling proteins [inhibitor of nuclear factor
κB α (IκBα) and various kinases] (Fig. 1, A
Immunoblotting (also known as Western blotting) combined with digital image analysis and B). RIPA buffer also extracted the cy-
can be a reliable method for analyzing the abundance of proteins and protein modifica- toskeletal and cytoskeleton-associated pro-
tions, but not every immunoblot-analysis combination produces an accurate result. I teins, actin and focal adhesion kinase (FAK).
illustrate how sample preparation, protocol implementation, detection scheme, and However, tubulin and intermediate filament Fig. 2. Posttranslational modifications can move protein into the
normalization approach profoundly affect the quantitative performance of immunoblot- proteins (lamin A and KRT5) showed sub- insoluble fraction of common lysis buffers. MCF10A-5E cells were
ting. This study implemented diagnostic experiments that assess an immunoblot-analysis stantial losses into the RIPA-insoluble frac- exposed to the Fas cross-linking agent anti-APO-1 (1 μg/ml) (48) for 24
workflow for accuracy and precision. The results showed that ignoring such diagnostics tion (Fig. 1C). Remarkably, RIPA insolubility hours, and then floating and adherent cells were lysed in NP-40 lysis buf-
can lead to pseudoquantitative immunoblot data that markedly overestimate or underes- was not limited to cytoskeletal proteins: the fer, RIPA buffer, or dithiothreitol-containing Laemmli sample buffer (SB).
timate true differences in protein abundance. transcription factor GATA2 and the cell-cell (A) Effect of solubilization conditions on the detection of cleavage (Clv.)
adhesion protein β-catenin were also pres- products of caspase-3. (B) Effect of solubilization conditions on the detec-
ent in the insoluble fraction. In contrast, ly-

A
tion of cleavage products of caspase-8. Vinculin, tubulin, GAPDH, Hsp90,
mong the most indispensible tools in cell tions of Towbin et al. (4) (25 mM tris, 192 sis with Laemmli sample buffer, followed by and p38 were used as loading controls where indicated. Data are repre-
signaling research is the immunoblot. mM glycine, 0.0375% SDS, and 10% methanol shearing of the viscous genomic DNA with a sentative of three experiments.
Fig. 1. RIPA buffer solubilizes many, but not all, cellular proteins. (A)
The premise of immunoblotting is sim- unless otherwise indicated); detection was high-gauge needle, solubilized proteins that
Examples of proteins that are entirely solubilized [100% in the superna-
ple, but execution is tricky, and there are performed on either a LI-COR Odyssey instru- are tightly associated with DNA, such as his-
tant (Sup)]. (B) Examples of proteins that are mostly solubilized (>90%
many variations in the method that can ment (for fluorescence detection) or a Bio-Rad tones (Fig. 1D). Despite rules of thumb for curacy and dynamic with an enhanced chemiluminescence (ECL)
Sup). (C) Examples of proteins that are partially solubilized (≤90% Sup).
affect the outcome (1). Add quantitation to the ChemiDoc MP gel imager (for chemilumines- protein solubility in various lysis buffers (6), range of immunob- cocktail (16) or a commercial substrate mar-
(D) Dimethyl-lysine 4 histone H3 (H3K4me2) resides almost entirely in the
end of an immunoblot and the complexity of cence detection); and quantitation of raw 16- these results show that it is best to confirm lot band intensities keted for high-sensitivity applications (file
RIPA-insoluble pellet (Pel). Band intensities were quantified from the 16-bit
implementations increases even further. Sur- bit digital images was implemented with the proper solubilization of proteins of interest is unknown. S2). Under the same immunoblotting condi-
digital image by densitometry in ImageJ and are shown normalized to the
prisingly, there are few objective studies on ImageJ gel analysis plug-in (5). Using film to before embarking on an immunoblot study. Using HT-29 cell tions, chemiluminescent exposures consis-
Sup lane for each target. n.d., not detected. Data are representative of two
quantitative immunoblotting in the primary perform quantitative immunoblotting was If 10 to 30% of a protein were consis- extracts, I performed tently yielded stronger band densities (Fig.
to four experiments.
literature (2, 3). Lacking a systematic assess- avoided entirely, because the dynamic range tently lost in the insoluble fraction, then an extended twofold 5, A to C). However, the linear dynamic range
ment of key factors, researchers are prone to of film is so small that quantitative analysis is the choice of lysis conditions would not be serial dilution from a was very limited, and signals often decreased
repeat or reinforce mistakes that others have virtually impossible (3). Film can make small critical. However, specific proteins can shift cultures of AC16 ventricular cardiomyocytes grossly overloaded sample (200 μg of extract) at high protein inputs (Fig. 5C). This can oc-
made before them. differences in abundance appear as large dif- between soluble and insoluble fractions in (15) and immunoblotted for multiple phos- to one below the limit of detection (100 ng of cur when side products of the peroxidase-cat-
Here, I analyze how various methodological ferences in band intensity. When saturated, a stimulus-dependent manner. As an ex- phorylation sites along the Akt–glycogen extract). Two replicate gels were transferred alyzed reaction are oxidized and precipitated,
choices affect the ability to perform quantita- film exposures can also hide sample-to-sam- ample, I activated the FAS death receptor synthase kinase-3 (GSK3)–glycogen synthase in buffer containing 10 or 20% methanol, and causing the membrane to “brown out” and
tive immunoblotting accurately and precisely. ple variations in high-abundance proteins in MCF10A-5E human breast epithelial cells (GS) signaling axis (Fig. 3, A to D). For these then membranes were probed for seven differ- absorb the emitted photons. Titrating down
The analysis revealed how seemingly minor such as loading controls. Therefore, through- (8) and lysed the cells in NP-40 buffer (lack- phosphoproteins, the addition of phospha- ent targets (file S1). For actin and p38, I found the amount of protein or primary antibody
variations affect immunoblot linearity and out this study, all data were acquired as digi- ing SDS and deoxycholate), RIPA buffer, or tase inhibitors was more critical in RIPA buf- that detection was linear up to 50 μg of total can avoid the problem, provided that the re-
reproducibility, yielding pseudoquantitative tal images. The diagnostic experiments shown Laemmli sample buffer. Although the cleav- fer than in the nondenaturing NP-40 buffer. protein, irrespective of the methanol concen- searcher is aware of it.
numbers that are not directly proportional here can be easily adapted for other hardware age of caspase-3 was readily detected in all The extent of sensitivity depended strongly tration (Fig. 4, A and B). This zero-intercept
Optimizing loading controls
to the input material. After background sub- and reagent configurations. three preparations (Fig. 2A), cleaved forms on the phosphorylation site, with Thr308 linearity is ideal, because band density is di-
traction, quantitative immunoblots should of caspase-8 were only detected in Laemmli of Akt, Ser21 of GSK3α, and Ser9 of GSK3β rectly proportional to input material without Arguably, the biggest source of confusion in
strive for zero-intercept linearity: y = bx, RESULTS sample buffer (Fig. 2B). Thus, quantitative showing greater lability than Ser473 of Akt the need to refer to a calibration curve. By quantitative immunoblotting is the role of
where y is the quantified band intensity, x is Sample preparation: A critical factor for measures of caspase-8 processing would re- and Ser641 of GS. These results collectively contrast, Hsp90 and tubulin showed a hyper- protein loading and loading controls (17). Nor-
the abundance of the protein or modification quire the Laemmli preparation for accurate showed that lysis buffer composition sub- bolic saturation under both transfer condi- malizing for cell numbers across samples is
quantitative immunoblotting
state in the sample, and b is a proportional- results (9–11). My laboratory has found that stantially affects the results of quantitative tions with less than 25 μg of total protein (Fig. challenging, because it is difficult to estimate
ity coefficient. The value of b is flexible, but The conditions of cell lysis have a profound similar precautions are required for moni- immunoblotting. 4, C and D). Saturation can result from steric changes in cell proliferation and death under
lines with nonzero intercepts indicate errors impact on the proteins that are extracted and toring regulated changes in intermediate- crowding of antibody epitopes, quenching of different conditions. Such estimates also do
Assessing immunoblot protocols by
in background subtraction, and nonlinear re- the condition in which they are preserved. For filament proteins (12), such as KRT5 (Fig. 1C). fluorescently labeled secondary antibodies, or not account for variations in initial seeding
serial dilution
lationships suggest problems with detection example, lysis of cells or tissues with purely Regulated oligomeric or polymeric protein oxidation of enzyme-conjugated secondary an- density and final lysate volumes, which will
sensitivity or saturation. Either scenario will nonionic detergents (Triton X-100 or NP-40) assemblies may be particularly susceptible to For immunoblotting, a single protocol that is tibodies. Regardless of the source, band densi- affect the observed protein abundance. Con-
yield fold change estimates that are skewed causes some proteins to partition into the differential partitioning between soluble and optimal for all electrophoresis transfer setups and ties in this regime no longer provide a linear sequently, immunoblot samples are often pre-
relative to the true differences among samples. soluble and insoluble (pellet) fractions after insoluble fractions. detection methods does not exist. However, the estimate of sample abundance, and calibra- pared according to total cellular protein (18,
Throughout this work, I systematically centrifugation. Radioimmunoprecipitation The stability and posttranslational modifi- quantitative accuracy and dynamic range of any tion is required to obtain accurate measure- 19), assuming that the average protein con-
altered several experimental parameters assay (RIPA) buffer—containing dilute SDS cations of lysate proteins are also affected by protocol can be assessed using a serial dilution ments. The improved transfer of proteins in tent per cell is constant across the different
that are often neglected or overlooked when (a denaturing detergent) and deoxycholate (a the activity of co-mingling cellular enzymes, of cell extract and a panel of primary anti- 20% methanol appeared to shift the detection conditions.
immunoblotting. Many other parameters disruptor of protein-protein interactions)—is such as proteases and phosphatases. These bodies. As an example, I sought to determine of multiple targets from a linear regime to one To complement total protein estimates, im-
were kept fixed: All gels were run as 15- widely used as a lysis buffer for whole-cell ex- enzymes are usually blocked with inhibitors wet-transfer conditions (specifically, metha- of hyperbolic saturation (Fig. 4, E to G). The munoblots typically include loading control
well, 1.5-mm-thick, tris-glycine minigels on traction. Nonetheless, RIPA buffer lysis still that are supplemented into nondenaturing nol concentration) that enabled quantitative results from this diagnostic study indicated proteins, which provide a secondary check
a Bio-Rad Protean III platform; all wet elec- generates an insoluble fraction with major lysis buffers, but the SDS and deoxycholate detection of most target proteins. Here, the that the lower methanol concentration was that roughly equal amounts of cellular mate-
trophoretic transfers were done onto low- protein constituents from the cytoskeleton in RIPA are sometimes assumed to inacti- transfer buffer always included 10% methanol preferred for my immunoblot protocol. rial have been added. Two key assumptions of
autofluorescence, 0.45-μm polyvinylidene and extracellular matrix (6). vate most cellular enzymes. I tested how ef- (see Materials and Methods), but other proto- Similar comparisons have also caused my the loading control are that (i) its abundance
difluoride (PVDF) under modified condi- To test how RIPA lysis conditions affected fectively RIPA and NP-40 buffers inhibited cols use 20% methanol according to the origi- laboratory to favor fluorescence-based de- is roughly constant across the different condi-
immunoblotting results, I lysed HT-29 hu- protein phosphatases by omitting from both nal conditions of Towbin et al. (4). Although tection over chemiluminescence whenever tions, and (ii) its immunoblot band intensity
Department of Biomedical Engineering, University of Virginia, man colon adenocarcinoma cells in RIPA buffers the Ser-Thr phosphatase inhibitor immunoblot bands are brighter and crisper possible. I compared the linearity of IRDye- is linearly reflective of its abundance. How-
Charlottesville, VA 22908, USA buffer, boiled the RIPA-insoluble pellet in microcystin-LR (13) and the Tyr phosphatase with the higher methanol concentration, how conjugated secondary antibodies to horserad- ever, a single loading control may not fulfill
*
Corresponding author. E-mail: kjanes@virgnia.edu an equal volume of dithiothreitol-containing inhibitor orthovanadate (14). I lysed parallel methanol percentage affects quantitative ac- ish peroxidase conjugates that were incubated both of these assumptions (2).

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W E S T E RN BLOT T ING : A G U I D E TO CU R R E N T M E TH O D S S E C T I O N T H R E E | RESEARCH ARTICLES

If total cellular protein can be quanti- Fig. 3. Phosphatase Fig. 4. Linearity and hyperbolic saturation
fied accurately, then total protein is loaded inhibitors are critical of immunoblots determined by serial dilu-
equivalently across samples, and one or two to preserve certain tion. (A and B) Immunoblots for actin and p38
loading controls suffice as a qualitative confir- phosphorylated resi- are linear under both transfer conditions. (C
mation of overall protein abundance (20–22). dues under certain and D) Immunoblots for Hsp90 and tubulin are
Conversely, if the total cellular protein is not lysis conditions. (A hyperbolically saturated under both transfer
known or cannot be determined accurately, and B) Effect of lysis conditions. (E to G) Linear detection of immu-
then the input must be normalized to some buffer and presence noblots for E-cadherin, ERK1/2 (extracellular
estimate of protein loading. A common ap- or absence of phos- signal–regulated kinases 1 and 2), and GAPDH
proach found in the literature is to normalize phatase inhibitors occurred with tank transfer conditions contain-
by only one loading control, but this scaling (PPIs) on the detec- ing 10% methanol. HT-29 cells were lysed in RIPA
is highly problematic. Taking one unknown tion of phosphorylated buffer, immunoblotted for the indicated targets,
quantity (the protein of interest) and divid- Akt (p-Akt) on Thr308 and imaged. Left: Immunoblots. Middle: Log-log
ing it by another unknown quantity (a single (T308) and Ser473 plots of the quantified band intensities from the
loading control) creates a number with very (S473). (C) Effect of blots on the left. Right: Linear plots of the same
poor statistical properties, including an un- lysis buffer and pres- data. Linear fits are gray when the hyperbolic
defined mean. The dangers of single-variable ence or absence of model is no better than the linear model for that
normalization have long been recognized in PPIs on detection of transfer condition. Linear fits are red when the
data from microarrays (23) and quantitative GSK3α/β phosphory- linear fit of the associated transfer condition is
polymerase chain reaction (PCR) (24), but not lated on Ser21 and Ser 9 better than the linear fit of the other transfer
in data from immunoblotting. A solution is to (p-GSK3α/β). (D) Ef- condition. Hyperbolic fits are green when the
aggregate the band intensities from multiple fect of lysis buffer and hyperbolic model is better than the linear model
loading controls, calculating a mean estimate presence or absence for that transfer condition. Data are in blue when
of total cellular content that is less sensitive of PPIs on detection neither the linear nor the hyperbolic model pro-
to the technical or biological fluctuations of a of GS phosphorylated vides a better fit. Model comparisons were done
single loading control (12, 25). on Ser 641 (p-GS). AC16 by the F test [false discovery rate (FDR) = 5%;
To demonstrate the utility of multiprotein cells were lysed in RIPA or NP-40 lysis buffer with or without PPIs. Vinculin, tubulin, GAPDH, and ac- n = 5 to 8 dilutions]. See file S1 for raw images
normalization, I immunoblotted for linker tin were used as loading controls where indicated. Total Akt, GSK3α/β, and GS were used to monitor and calculations.
phosphorylation of Smad2 on Ser245/250/255 (p- specific changes in protein abundance. Band intensities were quantified from the 16-bit digital image
Smad2 linker) in MCF10A-5E cells that had by densitometry in ImageJ and are shown normalized to the average +PPI conditions for each target by chemiluminescence, MemCode is not
been stimulated with transforming growth across both lysis conditions. Data are representative of two experiments. suitable for two-color fluorescence detection.
factor–β (TGFβ) (file S3). Under these condi- I uncovered a different set of problems with
tions, Smad2 linker phosphorylation is medi- ally no effect (Fig. 6C). This does not imply with decreased protein content for loading Ponceau S. Compared to MemCode, the red
ated by cyclin-dependent kinases (CDKs) (26), that GAPDH is always a good loading control controls (2). However, normalizing from dif- Ponceau S stain was not as efficiently detected
so cells were additionally pretreated with or or that tubulin is always a bad one; rather, it ferent lanes will miss lane-specific irregulari- by the CCD camera (fig. S1, A and D). How-
without the pan-CDK inhibitor flavopiridol emphasizes the danger of relying on a single ties in sample preparation or electrophoretic ever, its image densitometry was linear as a
(27). The TGFβ stimulation ± flavopiridol measured variable to estimate total protein transfer, which can be minimized with good function of lysate amount, and there was no
inhibition experiment was performed in bio- content. As higher-order combinations of technique but not eliminated. background fluorescence caused by Ponceau
logical quadruplicate to assess reproducibility loading controls were tested as normalizers, A possible alternative to multiple S staining or erasure (fig. S1, E to G). Pon-
of p-Smad2 linker quantification. Lacking any I found that the coefficient of variation of p- loading controls is to use reversible total ceau S appeared to fill all the requirements
total protein normalization, p-Smad2 linker Smad2 linker steadily improved toward 7 to protein stains that are compatible with for a total protein stain, except for one major
densitometry was variable, with flavopiridol 8%, consistent with values reported previ- PVDF membranes (such as MemCode or drawback—the zero intercept of its densitom-
producing only a marginally significant de- ously (10, 11). This reproducibility became Ponceau S). For a PVDF-compatible stain etry could not be accurately estimated from a
crease in phosphorylation (Fig. 6, A and B). less dependent on the specific combination to be useful, it must be quantitative for blank region of the PVDF membrane (fig. S1D,
To improve reproducibility, I blotted the same of loading controls (note the shrinking error total cellular protein (directly proportional lane B), causing a negative bias of ~10,000 in-
membrane for five potential loading controls: bars in Fig. 6C), indicating that I had con- or, at least, hyperbolically saturating), and tensity units (fig. S1E). Without a lysate cali-
total Smad2, tubulin, Hsp90, GAPDH, and verged on a measure of cellular content per it must not interfere with the subsequent bration curve to estimate this bias on a PVDF
p38. The antibodies for these proteins are lane that was truly representative. With all immunoblot. I tested two total protein stains membrane, I concluded that Ponceau S can-
from various hosts and yield single immuno- five loading controls, both the TGFβ-induced that can be reversibly applied to PVDF not be used for relative protein quantification.
reactive bands under the blotting conditions stimulation of p-Smad2 linker and its inhibi- membranes: MemCode Reversible Protein When immunoblotting for phosphoproteins,
used here. The properties of the antibodies tion by flavopiridol were highly significant Stain (commercially available from Thermo there are additional complications with eras-
and the detected control proteins enabled (Fig. 6D). Scientific) and Ponceau S (28). MemCode ing Ponceau S from PVDF membranes, be-
multiplex detection of loading controls to- Others have noted that many common yielded a strong blue banding pattern that cause the alkaline conditions for erasure will
gether with p-Smad2 linker or after a single loading controls are abundant proteins (for was readily detected by white-light epi- chemically modify phosphorylated Ser and
round of membrane stripping (see Materials example, tubulin) that result in saturated illumination and a charge-coupled device Thr residues (29).
and Methods). band intensities under the conditions needed (CCD) camera (fig. S1A). Using a blank region Given these data with the reversible total
Upon quantifying band densities in Im- to detect proteins or modification states of in- of the PVDF membrane to define a background protein stains, accurate quantitation of im-
ageJ, I compared the reproducibility of terest (2). On the basis of the results with the for subtraction, I found that total MemCode munoblot data should adopt the best prac-
p-Smad2 linker after normalization to all pos- serial dilutions (Fig. 4D), saturation is prob- band intensity increased hyperbolically tices of quantitative PCR (24) and use an
sible combinations of loading controls: 25 = ably the reason why tubulin worked poorly as with zero intercept over a relevant range assortment of three or more loading controls,
32 combinations. For single loading control a loading control for p-Smad2 linker in this of lysate amounts (fig. S1B). However, the spanning a range of abundances, when direct
normalization, the effect on replicate-to-rep- setting (Fig. 6B). However, in the context of staining procedure markedly increased the total protein measures are lacking. To mini-
licate reproducibility heavily depended on multiple loading controls that are averaged, 700-channel background fluorescence of the mize cost and effort, my laboratory detects
the choice of loading control. Normalizing to a saturated loading control negligibly affects membrane, and this background was not constitutively produced proteins with high-
GAPDH decreased the p-Smad2 linker coeffi- normalization because of its reduced sample- removed by the recommended stain removal affinity antibodies that work reliably at low
cient of variation by more than twofold (21 to-sample variation. An alternative strategy is (erasing) procedure (fig. S1C). Although concentrations (25 to 100 ng/ml), yield single
to 9%), but normalizing to tubulin had virtu- to prepare a separate set of immunoblot lanes potentially useful for immunoblots detected immunoreactive bands, and thus are ideal for

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multiplexing (see Materials and Methods; lines, I verified that loading change in protein; instead, they
Table 1). the same total mass of HT-29 can substantially underestimate
and AC16 extract gave approxi- it. To demonstrate, I plotted di-
Stripping, reprobing, and the total
mately equivalent band intensi- lutions of an unstimulated and
protein control
ties for several loading controls stimulated extract for a theo-
Aside from loading controls for total cel- (Fig. 8C). Hsp90 abundance retical immunoblot band that
lular content, immunoblots that quantify was higher (per total mass) in is hyperbolically saturated (Fig.
protein modification states should con- HT-29 cells, whereas vincu- 9). There is a clear threefold
tain an additional control: an immunoblot lin and tubulin were higher in change in abundance when the
for the total protein. This control serves to AC16 cells, and GAPDH and ac- target is immunoblotted under
gauge how much of the observed change tin were approximately equal. conditions where the linear ap-
in protein modification can be explained Fig. 5. Quantitative immunoblotting is challenging when imaging by chemiluminescence. (A to C) The uncorrelated abundance of proximation is accurate (<10
by differences in target abundance. For HT-29 lysates were prepared as in Fig. 4, immunoblotted for the indicated proteins, and imaged by IRDye these “housekeeping proteins” μg of extract in this example).
rapid experiments that are expected to fluorescence, ECL, or SuperSignal West Femto chemiluminescence as described (12, 16, 45, 46). Linear suggests that the cell extracts However, overloading the gel
avoid protein turnover and synthesis fits are shown in gray when the hyperbolic model is not significantly better than the linear model for that can be fairly compared on a with 50 μg of extract reduces
(such as the one shown in Fig. 6), the to- imaging condition. Linear fits are shown in red when the linear fit of the associated imaging condition is total protein basis. The results the difference to 1.4-fold. This
tal protein control can additionally con- significantly better than that of the other imaging conditions. Hyperbolic fits are shown in green when the further reinforced the impor- type of dampening has led some
tribute to a panel of loading controls if hyperbolic model is significantly better than the linear model for that imaging condition. Data are interpo- tance of using multiple loading to conclude that fluorescence
changes in band intensity are clearly co- lated in blue when neither the linear nor the hyperbolic model provides a better fit. All model comparisons controls. detection is not sensitive com-
fluctuating with other loading controls. were done by the F test at an FDR of 5% (n = 4 to 8 dilutions). See file S2 for raw images and calculations. To quantify the protein of in- pared to chemiluminescence
However, for comparisons on long time terest in cellular extracts, I ran (especially on film). However,
scales, across different cell types, or with artifact in the total ERK1/2 reprobe, with in- albumin (obtained as a calibrated standard the calibrated standards and the the comparison is not fair if the
rapid protein turnover, the total protein con- creased staining in the +EGF lane. The SDS from a protein assay kit), a standard curve extracts alongside one another 1.4-fold difference in antibody
trol is specifically important on its own and plus β-mercaptoethanol strip completely re- was constructed by running serially diluted and blotted with an antibody binding has been exaggerated
may not be a reliable indicator of loading. moved the GAPDH antibody and yielded the bovine serum albumin on a polyacrylamide that detects both the recombi- by a nonlinear chemilumines-
A common way to estimate target protein closest approximation of total ERK1/2 abun- gel alongside recombinant glutathione S- nant protein and the protein of cent reaction (Fig. 5, B and C).
abundance from a modification-specific im- dance, although with substantial loss of total transferase (GST)–tagged purified ERK2 and interest (Fig. 8, D to G). Note The analysis here also il-
munoblot is to strip the membrane of anti- protein from the membrane based on the im- p38 (22) (Fig. 8A). The gel was then stained that the ERK2 calibration can- lustrated that quantifying
body and then reprobe with an antibody that munoreactivity of the three additional load- with Coomassie blue and scanned for its near- not be used to quantify the Fig. 6. Reproducibility of quantitative immunoblots across biologi- phosphorylation-specific immu-
recognizes the total protein (or at least the un- ing controls (compare the glycine-stripped infrared fluorescence (34), yielding a digital ERK1 band at 44 kD because of cal replicates is improved after normalization to multiple loading noblots as a “phosphorylated-to-
modified form). Essential to this approach is membrane to the membrane stripped with image for densitometry in ImageJ (see Ma- differences in the immunoreac- controls. (A) Representative immunoblot for phosphorylated Smad2 on total ratio” is fraught with both
confirming that the stripping conditions have SDS plus β-mercaptoethanol). Moreover, after terials and Methods). To accommodate some tive epitope. In addition, a mass Ser245/250/255 (p-Smad2 linker) in MCF10A-5E cells stimulated with TGFβ (50 numerical complications (Fig. 6)
fully removed the original modification-spe- SDS plus β-mercaptoethanol stripping, the to- degree of band saturation and improve the correction is needed to account ng/ml) for 30 min with or without 1 hour of preincubation with 300 nM fla- and potential experimental ar-
cific antibody from the membrane of choice tal ERK1/2 bands still showed an artifactual dynamic range of detection, I fit the protein for the size difference between vopiridol. Tubulin, Hsp90, GAPDH, and p38 were used as loading controls. tifacts (Fig. 7). The ratio is fur-
(PVDF or nitrocellulose). If not, the reprobed increase in the EGF-treated sample. To avoid band intensities to a simple hyperbolic curve: the GST-tagged recombinant Total Smad2 was used to monitor overall changes in protein abundance ther prone to be misinterpreted
blot will show artifacts from the residual first the need to strip and reprobe the same blot, protein and the endogenous and served as a fifth candidate loading control for this analysis. (B) Raw as a phosphorylation stoichi-
antibody. As an example, I acutely stimulated an alternative is to use two-color fluorescence Intensity = a×Protein protein, because immunoblot- p-Smad2 linker densitometry quantified in ImageJ. (C) Decrease in the co- ometry, which cannot be calcu-
AC16 cells with epidermal growth factor (EGF) detection with phosphorylation-specific and b+Protein ting quantifies antigen indepen- efficient of variation among p-Smad2 biological replicates with increasing lated in experiments that use
for 5 min and immunoblotted for phosphory- total antibodies that are raised in different dently of its mass (the correction numbers of loading controls. The best (GAPDH) and worst (tubulin) single different antibodies to detect
lated ERK1/2 (p-ERK1/2) with an antibody hosts and against different epitopes (Fig. 7C). with two free parameters (a and b) that is 42/69 for ERK2 and 38/65 for loading control normalizations are highlighted. (D) p-Smad2 linker densi- the phosphorylated and total
that is difficult to remove. Technical replicates Another option is to measure total target pro- were estimated by least-squares regression. p38; Fig. 8, E and G). This analy- tometry after normalization to the mean band intensity of tubulin, Hsp90, protein because of differences
of the same two lysates showed good repro- tein by immunoblotting a replicate set of sam- The modeled fit captured all of the albu- sis showed that AC16 cells had GAPDH, p38, and total Smad2 for each biological replicate. For (B) and (D), in antibody affinity. Calculating
ducibility in the observed EGF-stimulated ples and confirming that the matched loading min standards and enabled mapping the greater than sevenfold more data are shown as means ± SE of n = 4 biological replicates, with differences phosphorylation stoichiometry
induction of p-ERK1/2, and loading controls controls are comparable to those in the modi- measured GST-ERK2 and GST-p38 band ERK2 protein compared to HT- in means assessed by Welch’s two-sided t test. For (C), data are shown as by immunoblotting requires
were consistent across the membrane (Fig. fication-specific immunoblot (Fig. 7D). densities to total protein amount (Fig. 8B). 29 cells, despite only about two- mean coefficients of variation ± SD of n = 1 to 10 possible normalization electrophoretic conditions that
7A). After cutting the membrane into thirds, I Dividing by the volume of sample loaded into fold difference in band intensity combinations for the indicated number of loading controls. See file S3 for separate the phosphorylated
Advanced quantitative immunoblotting:
tested three stripping conditions: (i) a gentle the gel resulted in an estimated concentra- (Fig. 8E). The limited difference raw images and calculations. and total forms by mobility (41).
Absolute quantification
low-pH glycine buffer solution [1.5% glycine tion of full-length protein in the recombi- in band intensity was due to the Modification stoichiometry can
(pH 2.2), 0.1% SDS, and 1% Tween], (ii) a more For some computational models of biochemi- nant preparation. saturation of the ERK2 immu- be critical under certain condi-
stringent guanidinium solution (6 M guani- cal networks, the absolute abundances of Size separation of the recombinant protein noblot at high protein concentrations, which noblotting? When chemiluminescence is tions (42), but there are also examples where
dine-HCl) (30), and (iii) a high-stringency SDS specific cellular proteins are needed (32, 33). before quantification is critically important, was captured by the calibration curve. ERK2 used cavalierly, there is a danger of wildly increases in total protein have affected sig-
plus β-mercaptoethanol solution with heat Such applications require explicit calibra- because purified preparations often contain abundance in AC16 cells was also high com- exaggerated claims. For example, a twofold nal flow (43). Considering these caveats, my
[62.5 mM tris (pH 6.8), 2% SDS, and 100 mM tion using proper absolute standards on the cleavage products that add to total protein pared to the abundance of p38, which had a change in Hsp90 abundance might appear conclusion is that phosphorylated-to-total
β-mercaptoethanol at 50°C] (31). The stripped same immunoblot. For the cellular proteins but are not immunoreactive. Commercial concentration that was an order of magni- as a fivefold change in the intensity of the ratios based on stripped and reprobed mem-
membranes were blocked identically and rep- of interest, appropriate standards are puri- vendors of recombinant protein may simply tude lower (Fig. 8G). Although laborious, this band on an immunoblot (compare the ECL branes should be avoided with immunoblot
robed for total ERK1/2 along with three addi- fied recombinant proteins, which have been quantify total protein (full length plus frag- type of absolute quantification can be done values for ~6 and ~3 μg in Fig. 5B). A two- data.
tional loading controls. Reprobed membranes calibrated against a purified protein of known ments that are not useful for calibration), systematically for multiple cellular proteins fold change in GAPDH abundance could be A major challenge in evaluating the im-
were imaged and displayed identically to em- mass. ultimately resulting in an overestimation of and provide new insight into signaling func- “quantified” as a >20-fold change on the munoblot data of others is that publications
phasize differences in the immunoblot signal To illustrate this process, I quantified the cellular protein. For calibration, one should tion (35). basis of band intensity (compare the ECL and manuscripts will often omit details on
detected (Fig. 7B). absolute masses of ERK2 and p38 (per 25 μg also remember that unknown samples must values for 25 and ~13 μg in Fig. 5C). This acquisition that are considered “routine.”
The experiment revealed that the low-pH of cellular extract) in HT-29 and AC16 cells fall between calibration samples that are DISCUSSION makes numerical results look very impres- Those details matter, because the evalua-
glycine strip was ineffective at removing the (file S4). Absolute quantification enabled well fit by the linear or hyperbolic curve. Just because we can put numbers on an im- sive, but the truth is still a twofold change tion criteria of an immunoblot detected by
antibodies. Indeed, “total” ERK1/2 looked ERK2-p38 comparisons within each cell line Samples that fall outside the calibration age does not imply that we should—a quan- in abundance. film are different than those of an immu-
identical to p-ERK1/2, and there was even re- as well as between cell lines. range will cause the linear or hyperbolic tified biomolecule should relate directly to With fluorescence detection, there is not noblot detected by fluorescence. When such
sidual GAPDH antibody staining. By contrast, Recombinant ERK2 and p38 proteins were equation to make extrapolations that could the true quantity of that biomolecule if it is the danger of a runaway reaction, but a per- information is missing, readers and review-
the guanidinium strip removed most of the cloned, purified, and quantified by protein be highly inaccurate. to be meaningful (8, 36–40). What are the sistent concern is saturation (Fig. 4). Satu- ers can distinguish a film exposure by the
GAPDH antibody but left a clear p-ERK1/2 assay. Using a known mass of bovine serum Before making comparisons between cell implications of pseudoquantitative immu- rated immunoblots do not overestimate a hazy gray of the background and the blurred

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cystin-LR (1 μg/ml), and 200 μM sodium or- away from their target epitopes with PBS
thovanadate], NP-40 buffer [50 mM tris-HCl buffers. In this circumstance, the 0.5× block-
(pH 8.0), 150 mM NaCl, 0.5% NP-40 substitute, ing buffer should be prepared with Odyssey
5 mM EDTA supplemented with aprotinin (10 blocking buffer (LI-COR; # 927-50000) and
μg/ml), leupeptin (10 μg/ml), pepstatin (1 μg/ diluted in an equal volume of tris-buffered
ml), 1 mM PMSF, microcystin-LR (1 μg/ml), saline (TBS) throughout the procedure. TBS
and 200 μM sodium orthovanadate], or di- buffers were also used for the fluorescence-
thiothreitol-containing Laemmli sample buf- chemiluminescence comparison (Fig. 5).
fer [62.5 mM tris-HCl (pH 6.8), 2% SDS, 10% All blocking steps used a surface-to-volume
glycerol, 100 mM dithiothreitol, and 0.01% ratio of 5-ml 0.5× blocking solution per 2
bromophenol blue]. Laemmli sample buffer ¾-inch × 3 ¼-inch membrane from a 15-well
lyses were performed at room temperature minigel. The membrane and 0.5× blocking
Fig. 7. Membrane stripping and reprobing is a quantitative trade-off between antibody removal and total protein loss. (A) Replicate immunoblots to avoid precipitating the SDS, and viscos- solution were sealed in a plastic bag and in-
for ERK1/2 phosphorylated on Thr202 and Tyr204 of ERK1 and Thr185 and Tyr187 of ERK2 (p-ERK1/2) in AC16 cells stimulated with EGF (100 ng/ml) for 5 min. ity of these lysates was reduced by passing cubated on a rotating platform for 1 hour at
GAPDH and tubulin were used as loading controls in the first immunoblot. (B) Reprobe of the membrane in (A) for total ERK1/2 after stripping with glycine them vigorously through a 25-gauge needle 5 room temperature. Fig. 9. Quantifying partially saturated im-
buffer, guanidinium, or β-mercaptoethanol (βME) stripping buffer. Vinculin, Hsp90, and actin were used as loading controls for the reprobed blots. (C) to 10 times. For RIPA and NP-40 lysates, pro- munoblots can markedly underestimate dif-
Immunoblotting: Antibody probing ferences between samples. In this theoretical
Two-color fluorescence immunoblot for p-ERK1/2 (green) and total ERK1/2 (magenta) of the same lysates as in (A). Vinculin and Hsp90 were used as tein concentrations were determined with
loading controls. (D) Direct immunoblot for total ERK1/2 of the same lysates as in (A). A lower percentage polyacrylamide gel was used in (C) and (D) to the bicinchoninic acid assay (Thermo Fisher After blocking, membranes were incubated example, a serial dilution is performed with un-
emphasize the total ERK1/2 upshift after stimulation with EGF. GAPDH, vinculin, Hsp90, and tubulin were used as loading controls. Data are representative Scientific). with 0.5× blocking solution + 0.1% Tween stimulated and stimulated extracts. The relative
of two experiments. For solubilization comparisons (Fig. 1), 20 containing primary antibodies recogniz- change in the linear range (b) of the immunob-
HT-29 cells were lysed in RIPA buffer supple- ing the proteins or epitopes listed in Table 1. lot is [99 (blue)/33 (red)] ~ 3-fold, whereas the
mented with protease and phosphatase in- All primary antibody steps used a surface- relative change at fivefold higher loading is only
hibitors as described above. After incubation to-volume ratio of 5-ml primary antibody 1.4-fold (36%).
on ice and centrifugation, the RIPA-insoluble solution per 2 ¾-inch × 3 ¼-inch membrane
pellet was boiled for 5 min in an equivalent from a 15-well minigel. The membrane and ary antibody solution were sealed in a plastic
volume of dithiothreitol-containing Laemmli primary antibody solution were sealed in a bag and incubated on a rotating platform for
sample buffer. plastic bag and incubated on a rotating plat- 1 hour at room temperature.
form overnight at 4°C. Membranes were removed from secondary
Immunoblotting: Polyacrylamide gel
Antibody pairs raised in different species antibody solution and washed on a rocking
electrophoresis
were routinely multiplexed when using two- platform for 4 × 5 min in ~25 ml of PBS-T.
Immunoblotting was performed as described color fluorescence detection. In addition, To remove residual Tween 20, which is highly
(12, 45, 46), but the details of the implemen- primary antibodies with negligible off-tar- autofluorescent upon 700-nm excitation, the
tation will be elaborated upon here. Samples get bands could be multiplexed in the same membrane was washed for 5 min in ~25 ml
were prepared in dithiothreitol-containing detection channel if the molecular weights of PBS before scanning. Fluorescence images
Laemmli sample buffer to a total volume of of their protein targets could be clearly re- were obtained on an Odyssey infrared scan-
20 or 40 μl. Polyacrylamide gels (8, 10, 12, or solved from one another. This single-color ner (LI-COR) at 169-μm resolution and 0-mm
15%) of 1.5-mm thickness were cast according multiplexing enabled concurrent detection focus offset. Fluorescence channel intensi-
to (1), and samples were electrophoresed in of multiple loading controls. Common pri- ties ranged from 5.0 to 8.5 depending on the
tris-glycine running buffer (25 mM tris base, mary single-channel combinations included immunoblot.
Fig. 8. Workflow for absolute protein quantification. (A) Serial dilution of an albumin standard to calibrate recombinant purifications of GST-ERK2 250 mM glycine, and 0.1% SDS) at 130 V until antibodies recognizing tubulin (50 kD) +
Immunoblotting: Chemiluminescence
and GST-p38 by Coomassie staining. (B) Albumin band intensity (black) plotted as a function of protein and fit to a hyperbolic model (gray) that infers the dye front reached the end of the gel. GAPDH (36 kD), Hsp90 (90 kD) + p38 (38
detection
the amounts of GST-ERK2 (green) and GST-p38 (purple) proteins. (C) HT-29 and AC16 cells have roughly equal protein constituents by mass based on kD), vinculin (120 kD) + actin (42 kD), and
Immunoblotting: Electrophoretic
the amount of Hsp90, vinculin, tubulin, GAPDH, and actin detected in 25 μg of each sample. (D) Serial dilution of the GST-ERK2 standard to calibrate en- vinculin (120 kD) + tubulin (50 kD) + GAPDH Membranes were removed from primary an-
transfer
dogenous abundances of ERK2 in HT-29 and AC16 cells. (E) GST-ERK2 band intensity (black) plotted as a function of protein input and fit to a hyperbolic (36 kD). tibody solution and washed on a rocking plat-
model (gray) that infers the amount of ERK2 in HT-29 cells (blue) and AC16 cells (red). (F) Serial dilution of the GST-p38 standard to calibrate endogenous Proteins from the polyacrylamide gel were form for 4 × 5 min in ~25 ml of TBS + 0.1%
Immunoblotting: Fluorescence
abundances of p38 in HT-29 and AC16 cells. (G) GST-p38 band intensity (black) plotted as a function of protein input and fit to a hyperbolic model (gray). transferred to a PVDF membrane (Milli- Tween 20 (TBS-T). After washing, membranes
detection
The model was used to infer the amount of ERK2 in HT-29 cells (blue) and AC16 cells (red). Data are representative of two experiments. See file S4 for raw pore; Immobilon-FL, 0.45-μm thickness) in were incubated with 0.5× blocking solution
images and calculations. a Mini Trans-Blot Electrophoretic Transfer Membranes were removed from primary anti- + 0.1% Tween 20 containing horseradish per-
Cell (Bio-Rad) under modified conditions of body solution and washed on a rocking plat- oxidase–conjugated goat anti-rabbit (Jackson
Towbin  et al. (4) (25 mM tris, 192 mM gly- form for 4 × 5 min in ~25 ml of PBS + 0.1% ImmunoResearch; #111-035-144, 1:10,000)
borders of bands resulting from the flatbed ity. There are straightforward ways to diag- from M. Davidson (Columbia University) and cine, 0.0375% SDS, and 10% methanol unless Tween 20 (PBS-T). After washing, membranes or anti-mouse (Jackson ImmunoResearch;
optical scan of a film at arbitrarily high reso- nose immunoblot accuracy (Figs. 4, 5, and maintained in Dulbecco’s modified Eagle’s otherwise indicated). Transfers were electro- were incubated with 0.5× blocking solution + #115-035-146, 1:10,000). All secondary anti-
lution (44). Digitally acquired immunoblots 8) and precision (Fig. 6). We should all be medium/F-12 medium (Life Technologies) phoresed at 100 V for 1 hour under ambient 0.1% Tween 20 + 0.01% SDS containing one body steps used a surface-to-volume ratio of
will often have a whiter background with encouraged to complete these diagnostics plus 12.5% tetracycline-free fetal bovine se- conditions with an ice block in the transfer or more of the following secondary antibod- 5-ml primary antibody solution per 2 ¾-inch
crisper bands that may appear pixelated on our own targets and immunoblot setups rum (Clontech) and penicillin-streptomycin tank and the transfer tank surrounded by ies: IRDye 800CW–conjugated goat anti- × 3 ¼-inch membrane from a 15-well mini-
because of binning on a CCD camera or the before diving in to generate “real data.” The (Gibco). ice. rabbit (LI-COR; #926-32211, 1:20,000), IRDye gel. The membrane and secondary antibody
step size of a fluorescence scanner. Although stakes are simply too high to do otherwise. Cells were stimulated with the indicated 800CW–conjugated goat anti-mouse (LI-COR; solution were sealed in a plastic bag and in-
Immunoblotting: Membrane blocking
some may find them less aesthetically ap- concentrations of anti-APO-1-3 cross-linking #926-32210, 1:20,000), IRDye 680–conju- cubated on a rotating platform for 1 hour at
pealing, digitally acquired images provide antibody (Axxora), TGFβ (PeproTech), or EGF After transfer, the molecular weight markers gated goat anti-rabbit (LI-COR; #926-32221, room temperature.
the more accurate representation of band MATERIALS AND METHODS (PeproTech) for the indicated times, washed on the membrane were overwritten with a 1:20,000), IRDye 680LT–conjugated goat Membranes were removed from secondary
intensity and its relationship to sample with ice-cold PBS (phosphate-buffered saline), lead pencil (to provide 700-channel fluores- anti-mouse (LI-COR; #926-68020, 1:20,000), antibody solution and washed on a rocking
Cell lines, stimulation, and lysis
abundance. and then lysed in RIPA buffer [50 mM tris-HCl cence), and the membrane was blocked with and IRDye 680LT–conjugated donkey anti- platform for 4 × 5 min in ~25 ml of TBS-T.
The message of this research resource HT-29 cells were obtained from the American (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.5% 0.5× blocking buffer: Odyssey blocking buf- chicken (LI-COR; #926-68028, 1:20,000). All To enable a fair comparison with fluorescence
is not that chemiluminescence cannot be Type Culture Collection and maintained as sodium deoxycholate, 0.1% SDS, 5 mM EDTA fer (LI-COR; #927-40000) diluted in an equal secondary antibody steps used a surface-to- detection, the membrane was washed for 5
quantitative or that film exposures are al- recommended. The 5E clone of MCF10A cells supplemented with aprotinin (10 μg/ml), leu- volume of PBS. Although not observed for the volume ratio of 5-ml primary antibody solu- min in ~25 ml of TBS before exposing. Mem-
ways inappropriate. Rather, I want to convey was isolated and maintained as previously peptin (10 μg/ml), pepstatin (1 μg/ml), 1 mM immunoblots here, some phosphorylation tion per 2 ¾-inch × 3 ¼-inch membrane from branes were covered with an ECL solution
that with numbers comes great responsibil- described (8). AC16 cells (15) were purchased phenylmethylsulfonyl fluoride (PMSF), micro- site–specific antibodies can be competed a 15-well minigel. The membrane and second- composed of 1.25 mM luminol, 2 mM 4-iodo-

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Table 1. List of antibodies used and proteins or epitopes detected. BD, BD ceau S–stained membranes were treated with A Laboratory Manual (Cold Spring Harbor 23. J. P. Brody, B. A. Williams, B. J. Wold, S. R. G. N. Hortobagyi, M. C. Hung, Cancer Res. 74,
Biosciences; CST, Cell Signaling Technology; SCBT, Santa Cruz Biotechnol- 0.1 N NaOH for 30 s and washed with running Laboratory Press, Cold Spring Harbor, NY, ed. Quake, Proc. Natl. Acad. Sci. U.S.A. 99, 12975– 4822–4835 (2014).
ogy; Thermo, Thermo Fisher Scientific; KLF4, Kruppel-like factor 4; MCL1, deionized water for 2 min. 4, 2012), p. 1890. 12978 (2002). 44. M. Gassmann, B. Grenacher, B. Rohde, J. Vogel,
myeloid cell leukemia 1; PDI, protein disulfide isomerase. For both total protein stains, digital images 2. S. C. Taylor, A. Posch, Biomed. Res. Int. 2014, 24. C. Tricarico, P. Pinzani, S. Bianchi, M. Electrophoresis 30, 1845–1855 (2009).
were captured on a ChemiDoc MP gel imager 361590 (2014). Paglierani, V. Distante, M. Pazzagli, S. A. Bustin, 45. S. S. Bajikar, C. Fuchs, A. Roller, F. J. Theis, K. A.
(Bio-Rad) with “Colorimetric” settings (2 × 2 3. S. C. Taylor, T. Berkelman, G. Yadav, M. C. Orlando, Anal. Biochem. 309, 293–300 Janes, Proc. Natl. Acad. Sci. U.S.A. 111, E626–
camera binning). Fluorescence images were Hammond, Mol. Biotechnol. 55, 217–226 (2002). E635 (2014).
obtained on an Odyssey infrared scanner (LI- (2013). 25. J. Vandesompele, K. De Preter, F. Pattyn, 46. L. Wang, J. S. Brugge, K. A. Janes, Intersection
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6. L. C. Ngoka, Proteome Sci. 6, 30 (2008). J. Yu, S. Fujisawa, A. Barlas, A. N. Miller, K. 48. B. C. Trauth, C. Klas, A. M. Peters, S. Matzku, P.
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and 10% glacial acetic acid on a rocking plat- (2010). 4270s–4275s (2004). ACKNOWLEDGMENTS
form for 1 hour at room temperature and then 9. K. A. Janes, J. G. Albeck, S. Gaudet, P. K. 28. O. Salinovich, R. C. Montelaro, Anal. Biochem. I thank D. Gioeli, M. Weber, and D. Brautigan for
destained for several hours with 30% metha- Sorger, D. A. Lauffenburger, M. B. Yaffe, Science 156, 341–347 (1986). comments on this manuscript; Z. Chitforoushzadeh
nol + 10% glacial acetic acid until the back- 310, 1646–1653 (2005). 29. G. Taborsky, Phosphoproteins. Adv. Protein and S. Bajikar for lysate preparations; M. Shah for
ground was acceptable. The stained gel was 10. S. Gaudet, K. A. Janes, J. G. Albeck, E. A. Pace, Chem. 28, 1–210 (1974). recombinant proteins; T. Allen and C. Smolko for
scanned on an Odyssey infrared scanner (LI- D. A. Lauffenburger, P. K. Sorger, Mol. Cell. 30. S. Gallagher, S. E. Winston, S. A. Fuller, J. G. MemCode Reversible Protein Stain; S. Gaudet for
COR) at 169-μm resolution and 0.5-mm focus Proteomics 4, 1569–1590 (2005). Hurrell, Curr. Protoc. Immunol. Chapter 8, Unit immunoblotting anecdotes; and C. Borgman for
offset for 700-channel fluorescence. 11. K. A. Janes, S. Gaudet, J. G. Albeck, U. B. 8.10 (2001). copyediting the manuscript. Funding: This work
Nielsen, D. A. Lauffenburger, P. K. Sorger, Cell 31. S. H. Kaufmann, C. M. Ewing, J. H. Shaper, was supported by the NIH (#1-R21-AI105970),
Image densitometry
124, 1225–1239 (2006). Anal. Biochem. 161, 89–95 (1987). the American Cancer Society (#120668-RSG-11-
Raw 16-bit TIFs (tagged image files) were 12. C. C. Wang,S. S. Bajikar, L. Jamal, K. A. Atkins, 32. O. E. Sturm, R. Orton, J. Grindlay, M. Birtwistle, 047-01-DMC), the Pew Charitable Trusts (#2008-
opened in ImageJ (5) and rotated to align K.A. Janes, Nat. Cell Biol. 16, 345–356 (2014). V. Vyshemirsky, D. Gilbert, M. Calder, A. Pitt, 000410-006), and The David and Lucile Packard
immunoblot bands horizontally in the win- 13. C. MacKintosh, K. A. Beattie, S. Klumpp, P. B. Kholodenko, W. Kolch, Sci. Signal. 3, ra90 Foundation (#2009-34710). Author contributions:
dow. The rectangle tool was then used to Cohen, G. A. Codd, FEBS Lett. 264, 187–192 (2010). K.A.J. conceived the work, performed all
select lanes containing the band of interest. (1990). 33. B. Schoeberl, C. Eichler-Jonsson, E. D. Gilles, G. immunoblots and quantitative analyses, and wrote
The width of the lane rectangle was drawn 14. G. Huyer, S. Liu, J. Kelly, J. Moffat, P. Payette, Müller, Nat. Biotechnol. 20, 370–375 (2002). the paper. Competing interests: K.A.J. is a paid
as wide as possible without causing overlap B. Kennedy, G. Tsaprailis, M. J. Gresser, C. 34. S. Luo, N. B. Wehr, R. L. Levine, Anal. Biochem. consultant for Cell Signaling Technologies.
with bands from adjacent lanes. The height Ramachandran, J. Biol. Chem. 272, 843–851 350, 233–238 (2006).
of the lane rectangle was drawn long enough (1997). 35. C. Kiel, E. Verschueren, J. S. Yang, L. Serrano, Submitted 29 September 2014
to get a sample of the local background sur- 15. M. M. Davidson, C. Nesti, L. Palenzuela,W. F. Sci. Signal. 6, ra109 (2013). Accepted 16 March 2015
rounding the band of interest. Lane profiles Walker, E.Hernandez, L. Protas, M. Hirano, 36. K. A. Janes, D. A. Lauffenburger, Curr. Opin. Final Publication 7 April 2015
were plotted with the gel analysis plug-in, and N. D. Isaac J. Mol. Cell. Cardiol. 39, 133–147 Chem. Biol. 10, 73–80 (2006). 10.1126/scisignal.2005966
background was subtracted by connecting the (2005). 37. K. A. Janes, D. A. Lauffenburger, J. Cell Sci. 126, Citation: K. A. Janes, An analysis of critical factors
background intensity profiles to the left (top) 16. C. Haan, I. Behrmann, J. Immunol. Methods 1913–1921 (2013). for quantitative immunoblotting. Sci. Signal. 8, rs2
phenylboronic acid, and 0.0162% H2O2 (16). above. For the SDS plus β-mercaptoethanol and the right (bottom) of the band of interest 318, 11–19 zz(2007). 38. K. A. Janes, J. G. Albeck, L. X. Peng, P. K. Sorger, (2015).
Alternatively, membranes were covered with strip, membranes were incubated with by using the line tool. Last, the magic-wand 17. E. Marcus, Cell 159, 965–966 (2014). D. A. Lauffenburger, M. B. Yaffe, Mol. Cell.
SuperSignal West Femto reagent according SDS plus β-mercaptoethanol solution [62.5 tool was used to calculate the integrated area 18. B. J. Olson, J. Markwell, Curr. Protoc. Protein Proteomics 2, 463–473 (2003). SUPPLEMENTARY MATERIALS
to the manufacturer’s instructions (Thermo mM tris (pH 6.8), 2% SDS, and 100 mM within the band profile of interest and obtain Sci. Chapter 3, Unit 3.4 (2007). 39. M. D. Shults, K. A. Janes, D. A. Lauffenburger, B. www.sciencesignaling.org/cgi/content/full/8/371/
Fisher Scientific). Chemiluminescent expo- β-mercaptoethanol] and incubated in a dry- the final raw densitometry value. 19. P. K. Smith, R. I. Krohn, G. T. Hermanson, A. K. Imperiali, Nat. Methods 2, 277–284 (2005). rs2/DC1
sures were captured on a ChemiDoc MP gel air oven at 50°C for 30 min with occasional Mallia, F. H. Gartner, M. D. Provenzano, E. K. 40. L. Wang, K. A. Janes, Nat. Protoc. 8, 282–301 Fig. S1.Challenges with using total protein stains for
Recombinant protein purification
imager (Bio-Rad) with “Chemi Hi Resolution” agitation by hand. Stripped membranes were Fujimoto, N. M. Goeke, B. J. Olson, D. C. Klenk, (2013). normalization of quantitative immunoblots.
settings (2 × 2 camera binning). Exposure washed 3 × 5 min in ~25 ml of PBS before Recombinant GST-ERK2 and GST-p38 were Anal. Biochem. 150, 76–85 (1985). 41. J. G. English, J. P. Shellhammer, M. Malahe, P. File S1. Raw 16-bit images and densitometry
times were set manually to fill the bit depth blocking and immunodetection as described prepared by glutathione affinity chromatog- 20. K. J. Jensen, F. S. Garmaroudi, J. Zhang, J. Lin, C. McCarter, T. C. Elston, H. G. Dohlman, Sci. calculations from Fig. 4.
of the CCD camera without saturating any above. raphy in RIPL cells (Stratagene) as described S. Boroomand, M. Zhang, Z. Luo, D. Yang, H. Signal 8, ra5 (2015). File S2. Raw 16-bit images and densitometry
binned pixels. (22). Luo, B. M. McManus, K. A. Janes, Cell Host 42. O. Stemmann, H. Zou, S. A. Gerber, S. P. Gygi, calculations from Fig. 5.
Total protein staining after
Microbe 13, 67–76 (2013). M. W. Kirschner, Cell 107, 715–726 (2001). File S3. Raw 16-bit images and densitometry
Immunoblotting: Stripping and electrophoretic transfer Statistical analysis
21. B. H. Kang, K. J. Jensen, J. A. Hatch, K. A. Sci. 43. Y. H. Hsu, J. Yao, L. C. Chan, T. J. Wu, J. L. Hsu, calculations from Fig. 6.
reprobing
PVDF membranes were stained for total Serial dilutions were fit to linear or hyperbolic Signal. 6, rs13 (2013). Y. F. Fang, Y. Wei, Y.Wu,W. C. Huang, C. L. Liu, File S4. Raw 16-bit images and densitometry
For the low-pH glycine strip, membranes protein with MemCode Reversible Protein models by least-squares regression in IGOR 22. A. K. Bose, K. A. Janes, Mol. Cell. Proteomics Y. C. Chang, M. Y. Wang, C. W. Li, J. Shen,M. K. calculations from Fig. 8.
were incubated with low-pH glycine buffer Stain (Thermo Scientific) according to the Pro (WaveMetrics). The χ2 statistic from each 12, 797–806 (2013). Chen, A. A. Sahin, A. Sood, G. B. Mills, D. Yu,
solution [1.5% glycine (pH 2.2), 0.1% SDS, manufacturer’s recommendations. Erasure model fit was used together with the number
and 1% Tween] for 2 × 10 min at room of the MemCode stain was performed with of data points and the number of fitted pa-
temperature on a rocking platform. The the Eraser/Methanol solution for 20 min. rameters (one for the linear model and two
stripped membranes were washed 2 × 10 For total protein staining with Ponceau S, for the hypergeometric model) to calculate an
min in ~25 ml of PBS before blocking and membranes were incubated with 0.1% (w/v) F statistic that compares goodness of fit be-
immunodetection as described above. For Ponceau S in 5% acetic acid for 5 min and tween models. To correct for multiple-hypoth-
the guanidinium strip, membranes were washed 2 × 5 min in 10% acetic acid, followed esis testing, FDRs were calculated according
incubated with 6 M guanidine-HCl for 10 min by washes of 5 min in 100% methanol and 5 to Benjamini and Hochberg (47).
at room temperature on a rocking platform, min in a 70:30:4 volume ratio of methanol,
followed by a 5-min wash with PBS before acetic acid, and PEG-400 (polyethylene gly- REFERENCES AND NOTES
blocking and immunodetection as described col, molecular weight 400). For erasing, Pon- 1. M. R. Green, J. Sambrook, Molecular Cloning:

20 sciencemag.org SCIENCE SCIENCE sciencemag.org 21


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A neuronal antigen in the brains of were thioflavine-positive were


not positive for Alz-50 (Fig. 3, A
in native form on a Sepharose
6B column. Temporal cortex

Alzheimer patients
and B). The staining of plaques homogenate from Alzheimer
by Alz-50 was also studied by patients or normal subjects was
this method. All plaques bound centrifuged at 27,200g for 20
both Alz-50 and thioflavine. minutes, and the supernatant
Benjamin L. Wolozin, Alex Pruchnicki, Dennis W. Dickson, Peter Davies In addition, these results con- was run through the Sepharose
firmed the neuritic nature of 6B column. An immunoreac-
A monoclonal antibody was prepared against pooled homogenates of brain tissue from the antibody staining; staining tivity profile was obtained by
patients with Alzheimer’s disease. This antibody recognizes an antigen present in much was present in the neuritic pe- diluting each fraction 1:100 in
higher concentration in certain brain regions of Alzheimer patients than in normal brain. riphery of the plaques but ab- water, drying 50 µl of diluted
The antigen appears to be a protein present in neurons involved in the formation of sent in the amyloid core. fraction onto polyvinyl plates,
neuritic plaques and neurofibrillary tangles, and in some morphologically normal neurons Simple biochemical experi- and assaying by ELISA. The
in sections from Alzheimer brains. Partial purification and Western blot analysis revealed Fig. 1. Quantitation of Alz-50 reactivity in ments suggest that the Alz-50 profile from Alzheimer brain
the antigen from Alzheimer brain to be a single protein with a molecular weight of 68,000. temporal cortex of patients that had died of antigen is distinct from PHF, Fig. 2. (A) Immunocytochemistry of Alz-50 staining in the pyramidal revealed a single immunoreac-
Application of the same purification procedure to normal brain tissue results in the detec- Alzheimer’s disease and normal individuals. the major tangle component. layer of hippocampus from a brain affected by Alzheimeres disease. tive peak at the void volume.
tion of small amounts of a protein of lower molecular weight. Alzheimer’s disease cases were typical in both Unlike PHF, the Alz-50 anti- Vibra tome sections (40 µm) were cut from Formalin-fixed brain. The tis- By contrast, no immunoreac-
clinical and neuropathologic features (3). Brains gen is largely soluble in 0.01M sue was washed twice in TBS, incubated for 30 minutes in 0.25% Triton tivity was seen in the column
were obtained from normal individuals dying in tris-buffered saline (TBS) and X-100- TBS, washed once in TBS, and incubated for 30 minutes in dried fractionation of normal brain

A
lzheimer’s disease is a neurodegenera- gated goat antibody to mouse immunoglobu- hospital from lung or heart disease. These pa- completely soluble in TBS con- miLk (blocking solution) for 1 hour at room temperature to prevent non- at this dilution. At a lesser di-
tive disorder characterized clinically lins. Those antibodies that showed greater tients were not demented prior to death and had taining 5% sodium dodecyl specific binding of antibody. Alz-50 was diluted 1:5 in blocking solution and lution, normal brain immuno-
by progressive loss of intellectual func- than a 50% increase or decrease in binding no history of neurologic or psychiatric disease; sulfate (TBS-SDS) (6). Solubil- incubated with the tissue sections overnight at 4°C. Unbound antibody was reactivity was seen at the void
tion. This impairment of function ap- to homogenates of Alzheimer brain relative neuropathologic studies failed to reveal any sig- ity was tested by vortexing Al- removed by washing in TBS. Peroxidase coupled goat antibody to mouse volume. This result supports
pears to be correlated with numbers of to normal tissue were studied further. One of nificant pathology. The reactivity in the brains of zheimer cortex homogenate for immunoglobulin G was diluted 1:100 in blocking solution and incubated with the quantitative data shown in
neuritic plaques in the neocortex and with these antibodies, Alz-50, is described below. the Alzheimer patients is estimated to be 15 to 2 minutes in TBS or TBSSDS. the tissue sections for 1 hour. Antibody was visualized by incubating the sec- Fig. 1 demonstrating 15 to 30
loss of presynaptic markers of cholinergic Initial assays showed that the binding of 30 times greater than in the brains of the normal The homogenate was then tions for 8 minutes in a 0.1M tris solution, pH 7.4, containing 0.45 mg/ml times as much inimunoreactiv-
neurons (1). Neuritic plaques are composed Alz-50 was highly selective for brain tissue subjects. Various amounts of antigen (x axis) centrifuged at 10,000g for 10 of diaminobenzidine and 0.44 miM hydrogen peroxide. The tissuewas then ity in the Alzheimer brain as in
of degenerating axons and nerve terminals, from Alzheimer patients. Figure 1 shows that was dried onto 90-well polyvinyl plates (NUNC, minutes. Supernatant and pel- washed in TBS, dehydrated, and mounted. Magnification x 12.5. (B) Alz-50 the normal brain.
often surrounding an amyloid core and usu- 0.33 µg of temporal cortex homogenate from Germany). Nonspecific binding of protein to the let were separated, and the staining in hippocampus from a normal brain. (C) A plaque from frontal cor- Samples of supernatant from
ally containing reactive glial elements (2). Alzheimer patients gave an optical density antigen was blocked by incubating the plates pellet was washed twice by tex of a brain affected with Alzheimer’s disease. Magnification x25. Alzheimer and normal cor-
Another characteristic pathologic feature only slightly lower than 10 µg of temporal with 0.01M TBS, pH 7.4, plus 5% dried milk for 1 homogenizing and centrifug- tex were then studied by the
of Alzheimer’s disease, the neurofibrillary cortex homogenate from normal patients. hour. Alz-50 was diluted 1: 5 in blocking solution ing as above. The supernatant Western blot technique (Fig. 4).
tangle, is an intraneuronal mass composed From these data we conclude that the anti- and incubated overnight at 4°C. Unbound anti- and pellet were homogenized The major band in Fig. 4, lane
of normal intermediate filaments and paired gen is elevated 15 to 30 times in the temporal body was removed by washing five times with in water and various amounts A, has an apparent molecular
helical filaments (PHF) with unusual proper- cortices of the Alzheimer patients. Alz-50 re- 0.02% Tween-TBS. Peroxidase-coupled goat an- of each sample (10, 3, 1 µg per weight of 68,000 (68K). In Fig.
ties (3). activity was similarly elevated in the nucleus tibody to mouse immunoglobulins (Kirkegaard 50, µl) were dried onto poly- 4, lane B, the reactivity from
In studying the topographic distribution basalis and hippocampus. These areas, cor- & Perry) was diluted 1:100 in blocking solution, vinyl plates. The presence of the normal void volume frac-
of plaques and tangles in the brains of Al- tex, nucleus basalis, and hippocampus, are added to the plates, and incubated for 1 hour antigen was determined by an tion was brought out by load-
zheimer patients, we noted that the lesions all known to contain neuritic plaques and at room temperature. After five washes with enzyme-linked immunosorbent ing onto the polyacrylamide gel
occur with high frequency in regions receiv- neurofibrillary tangles in brains of patients 0.02% Tween-TBS, reactivity was visualized with assay (ELISA). PHF reactivity three times as much protein as
ing cholinergic innervation from the ventral with Alzheimer’s disease. Brain areas less af- 2,2’-azino-di’-3-ethyl-benzthiazoline solution was monitored by means of was used for the Western blot
forebrain (4). This cholinergic cell group fected by the disease, such as caudate, thala- (ABTS) (Kirkegaard & Perry). Results are ex- an antibody to PHF, antibody of the Alzheimer void volume
appears to be extremely vulnerable to the mus, or cerebellum demonstrated little or no pressed as means, with standard deviations indi- 704.1 (10). Alz-50 reactivity was Fig. 3. (A) Immunocytochemistry of Alz-50 in hippocampus from a brain fraction. The major band has
disease process, and evidence that cholin- reactivity. cated. Numbers of cases were ten for Alzheimer found in the TBS supernatant affected by Alzheimer’s disease. Arrows point to neurons that react with an apparent molecular weight
ergic nerve terminals participate in plaque The immunocytochemistry of Alz-50 on and six for normal. and was quantitatively recov- Alz-50 (A) but do not stain with thioflavine S (B). For details on the immuno- of 59K. A doublet at 245K is
formation has been presented (5). To better Formalin-fixed tissue was dramatic and was ered in the TBS-SDS superna- cytochemistry see legend to Fig. 1. The peroxidase reaction was developed also present.
define the relation between ventral forebrain consistent with the hypothesis that Alz-50 50, and reactivity was visualized with the use tant, whereas PHF reactivity by using 0.2 mg/ml of 4-chloronaphthol instead of diaminobenzidine (as in These results show that in
cholinergic neurons and the lesions of the was highly selective for neuronal compo- of peroxidase conjugated goat antibody to remained in the pellet after Fig. 2). Microscopy was performed by using phosphatebuffered saline (PBS)- TBS the antigen recognized
Alzheimer brain, we have prepared mon- nents in Alzheimer brain (Fig. 2A). Many mouse immunoglobulins. 4-Chloronaphthol TBS-SDS extraction. Thus, Alz- glycerol instead ofdehydration. Magnification x 12.5. (B) Thioflavine S histo- by Alz-50 is either aggregated
odonal antibodies to homogenates of ventral neurons in the pyramidal layer of the hippo- was used to visualize the peroxidase reac- 50 immunoreactivity is soluble chemistry of the same section from (A). Note the absence of thioflavine S or is part of a large complex.
forebrain tissue taken at autopsy from four campus were stained. The antigen was pres- tion; this compound, a peroxidase substrate, and segregates away from PHF staining of neurons (arrows). To remove the 4- chloronaphthol the section When dissociated, the epitope
patients with Alzheimer’s disease. The re- ent in cell bodies and in neurites. Figure 2C yields a product that precipitates in aqueous immunoreactivity. was dehydrated, whereupon the 4-chloronaphthol dissolved in the xylene, occurred as a single 68K pro-
sulting antibodies were screened on the basis shows that plaques were strongly stained by solution but is soluble in organic solvents. Enzyme experiments con- and then subsequently rehydrated. The section was then incubated in a tein that was distinctly differ-
of their ability to differentiate brain tissue the antibody. The staining was confined to The tissue section was photographed and the firmed this result. Before we 0.01% thioflavine S solution in Formalin and rinsed by dipping three times in ent from the 59K antigen from
from patients with Alzheimer’s disease and the neuritic meshwork present in plaques. 4-chloronaphthol was removed by dehydra- dried the Alzheimer brain 80% ethanol (fluorescence microscopy; magnification x 12.5.) normal brain. Several relevant
from normal subjects in both immunochemi- Darkly stained neurons and plaques were tion and xylene treatment. Finally, plaques homogenates onto polyvinyl proteins have subunit molecu-
cal and immunocytochemical procedures. found throughout Alzheimer hippocampus and tangles were stained with thioliavine S, plates, we treated them for 0, lar weights in the 68K range: in
Antibodies were initially assayed ac- and cortex. By contrast, there was virtually a sensitive histologic reagent for the demon- 20, or 60 minutes with trypsin or alkaline phorylated, although it is of interest because the cytoskeletal family there are neurofila-
cording to their ability to bind to brain ho- no staining of normal brain (Fig. 2B). This stration of these lesions, and the section was phosphatase. An ELISA was used to mea- many antibodies to neurofilament epitopes ment and tau proteins (7), in the cholinergic
mogenate that had been immobilized onto pattern of specificity was observed in a total photographed again. sure the sensitivity of the AIz-50 epitopes that identify neurofibrillary tangles fail to family there is choline acetyltransferase (8).
polyvinyl plates (1, µg per 50-mm diameter of eight brains from Alzheimer patients and Comparison of the staining patterns (Fig. to the treatment. Unlike the PHF, the Alz-50 react after phosphatase treatment. In addi- The Alz-50 antigen is therefore unlikely to be
well) by drying at 37°C for 1 hour. Antibody five brains from normal subjects. 3) revealed that many neurons were stained epitope was highly sensitive to trypsin. This tion, this result suggests that phosphoryla- neurofilament, tau protein, or choline acetyl-
binding was detected with peroxidase-conju- To determine the relation between Alz-50 both by the antibody and by thioflavine supports the hypothesis that the Alz-50 anti- tion of the epitope does not account for the transferase, because none of these proteins
staining of neurons and the presence of neu- S. However, several neurons were darkly gen is not PHF, and also demonstrates that ability of the antibody to distinguish be- seems to be elevated in concentrations 15 to
Deparnments of Pathology and Neuroscience, Albert rofibrillary tangles we used a double staining stained by the antibody and did not appear to the antigen is a protein. The AIz-50 epitope tween Alzheimer and normal brain. 30 times in the brains of Alzheimer patients.
Einstein College ofMedidne, 1300 Morris Park Avenue, technique. Vibratome sections of Formalin- contain neurofibrillary tangles. A small frac- is not phosphatase sensitive; this does not A Western blot of the AIz-50 antigen was Further, if the 68K Alzheimer type protein
Bronx, NY 10461. fixed Alzheimer tissue were reacted with Alz- tion of neurons that contained tangles and prove that the Alz-50 antigen is not phos- obtained by first purifying the Alz-50 antigen is related to the 59K protein, then the Alz-

22 Originally published 2 May 1986 in SCIENCE sciencemag.org SCIENCE SCIENCE sciencemag.org 23


W E S T E RN BLOT T ING : A G U I D E TO CU R R E N T M E TH O D S S E C T I O N T H R E E | RESEARCH ARTICLES

Fig. 4. Western blot of (A) Alzheimer and


(B) normal temporal cortex fractions from
REFERENCES AND NOTES
1. D. M. Bowen, C. B. Smith, P. Whitc, A. N. Serological analysis of a subgroup of male controls. Sera from the same patients
were also tested by the enzyme-linked

human T-lymphotropic retroviruses


the void volume of a Sepharose 6B column; Davison, Brain, 459 (1976); P. Davies, Brain immunosorbent assay (ELISA) with purified
(C) prestained molecular weight standards Res. 171, 319 (1979); P. Davies and A. J. F. HTLV-III as part of a larger, systematic
(Bethesda Research Laboratories). Temporal Maloney, Lancet 1976-H, 1403 (1976); E. K. serologic study of the prevalence of
cortex (2.5 g) was homogenized into 10 ml of
PBS and centrifuged at 20,000g for 20 minutes.
Perry, P. H. Gibson, G. Blessed, R. H. Perry,
B. G. Tomlinson, J. Neurol. Sci. 34, 247 (HTLV-III) associated with AIDS antibodies to HTLV-III in AIDS and pre-
AIDS patients (19).
The supernatant was run through a Sepharose (1977); G. Blessed, B. G. Tomlinson, M. Roth, Representative results are shown in Fig.
6B column and the void volume was collected. Br. Psychatry 114, 797 (1968). Jörg Schüpbach, Mikulas Popovic, Raymond V. Gilden, Matthew A. Gonda, 1. Sera from patients with AIDS or pre-
Protein determinations were performed and 2. R. D. Terry, N. K. Gonatas, M. Weiss, Am. J. M. G. Sarngadharan, and Robert C. Gallo AIDS, and from some homosexuals and
the samples were run on a 10% SDS-PAGE gel. Pathol. 44, 269 (1964). heroin-addicts, recognized a number of
Fifteen micrograms of protein was in the Al- 3. H. M. Wiseniewski, R. D. Tcrry, A. Hirano, J. The two main subgroups of the family of human T-lymphotropic retroviruses (HTLV) that specific antigens not detected by sera from
zheimer sample (A) and 45 µg of protein was in Neuropathol. Exp. Neurol. 29, 163 (1970). have previously been characterized are known as HTLV-I and HTLV-II. Both are associ- heterosexual subjects. The most prominent
the normal sample (B). The protein was trans- 4. M. M. Mesulam, E. J. Mufson, A. L. Levey, B. ated with certain human leukemias and lymphomas. Cell surface antigens (p61 and p65) reactions were with antigens of the following
ferred to nitrocellulose for 3 hours at 125 mA; H. Wainer,J. Comp. Neurol. 214, 170 (1983). encoded by HTLV-I are frequently recognized, at low titers, by antibodies in the serum of molecular weights: 65,000, 60,000, 55,000,
the buffer contained 19.2 mM glycine, 2.5 mM 5. R. G. Struble, L. C. Cork, P. Whitehouse, D. L. patients with acquired immunodeficiency syndrome (AIDS) or with signs or symptoms 41,000, and 24,000. Antigens with molecular
trizma base, and 20% methanol at pH 8.3. The Price, Science 216, 413 (1982); D. L. Price et that precede AIDS (pre-AIDS). This suggests an involvement of HTLV in these disor- weights of approximately 88,000, 80,000,
antibody reaction was developed as in Fig. 2, al., Neurosci. Comment. 1, 84 (1982). ders. Another subgroup of HTLV, designated HTLV-III, has now been isolated from many 39,000, 32,000, 28,000, and 21,000 gave less
except that phosphatase-coupled antibody 6. D. J. Selkoe, Y. Ihara, F. J. Salazar, Science patients with AIDS and pre-AIDS. In the studies described in this report, virus-associated prominent reactions. The reaction with the
was used instead of peroxidase-coupled an- 215, 1243 (1982). antigens in T-cell clones permanently producing HTLV-III were subjected to biochemical antigen of 55,000 (p55) only occurred in
tibody. Color development was achieved by 7. F. C. Chiu, W. T. Norton, K. L. Fields, J. and immunological analyses. Antigens of HTLV-III, specifically detected by antibodies sera that also recognized p24, suggesting a
means of BCIP/NBT (Kirkegaard & Perry). Neurochem. 37, 147 (1981). in serum from AIDS or pre-AIDS patients and revealed by the Western blot technique, relationship between the two antigens.
Omission of the incubation with Alz-50 com- 8. A. J. Levcy, D. B. Rye, B. H. Wainer, ibid. 39, are similar in size to those found in other subgroups of HTLV. They include at least three The specificity of these reactions was
pletely abolished staining. 1652 (1982). serologically unrelated antigenic groups, one of which is associated with group-specific studied by comparing Iysates of H4/ HTLV-
9. D. W. Dickson, Y. Krcss, A. Crowe, S.-H. Yen, antigens (p55 and P24) and another with envelope-related (p65) proteins, while the anti- III and HI7/HTLV-1II with Iysates of the
Am. J. Pathol. 120, 292 (1985). gens in the third group are of unknown affiliation. The data show that HTLV-III is clearly same cell clones, H4 and H17, before viral
distinguishable from HTLV-I and HTLV-II but is also significantly related to both viruses. infection (Fig. 2A). No antigen from the
50 antigen is unlikely to be neurofilament, minals in plaques and in most neurons with ACKNOWLEDGEMENTS HTLV-III is thus a true member of the HTLV family. uninfected clones reacted with the sera,
tau protein, or choline acetyltrarsferase. tangles. The surprising finding is that Alz- GM7288 from the National Institute of General with the exception of a protein with a

M
The relation among the proteins detected is 50 immunoreactivity appears to precede the Medical Sciences, The Mcknight Foundation, embers of the family of human lym- of blood transfusion recipients who later molecular weight of 80,000 in HI7 which
questionable: the relatively large amounts deposition of neurofibrils and PHFs to form The Joyce Mertz-Gilmore Foundation, and the photropic retroviruses (HTLV) have developed AIDS and of their blood donors bound antibodies from all of the human
of samples of normal brain tissue needed tangles. Thus Alz-50 may recognize a precur- Commonwealth Fund. We thank S.-H. Yen for the following features in common: a have revealed the presence, in the blood of serum samples tested (see Fig. 1B) but not
to detect any reactivity raises doubts about sor to tangle formation. Alternatively, Alz-50 antibody 704.1 to PHF and M. Scharff and E. pronounced tropism for OKT4+ lym- the donors, of antibodies to a retrovirus of from rabbit or goat serum. Antigens newly
the specificity of the antibody binding. Some immunoreactive neurons may be affected Fischberg for advice and assistance. We also phocytes (1), a reverse transcriptase the HTLV family (14). These findings suggest expressed after viral infection and recognized
monoclonal antibodies to tangles react with by Alzheimer pathophysiology but may not acknowledge use of the hybridoma facility of the (RT) with a high molecular weight (100,000) an involvement of viruses of the HTLV by the human serum used for this analysis
proteins in the 59K to 68K range (9), but form classical tangles. The biochemical data cancer center (CA 13330) at the Albert Einstein and a preference for Mg2+ as the divalent cat- family in the cause of AIDS and pre-AIDS. included p65, p55, p41, p39, p32, and p24.
none of these show quantitative differences showing that Alz-50 is not PHF antigen is College of Medicine. ion for optimal enzymatic activity (2, 3), and An involvement of HTLV-I alone appeared A large proteiri with a molecular weight
between normal and Alzheimer brain. The consistent with this discordance between the capacity to inhibit T cell function (4) or, doubtful, however, because antibody titers of approximately 130,000 and a protein of
points raised above suggest that the epitope the presence of Alz-50 and the presence of 30 October 1985; accepted 18 February 1986 h in some cases, kill T cells (5). Many HTLV to gp6l of HTLV-I in these patients are 48,000 were also detected. With this serum,
recognized by Alz-50 is a novel antigen. neurofibrillary tangle. The function and Alz-50 completely abolished staining. also have the capacity to transform infected generally very low and antibodies to the p55 consistently appeared as a doublet of
Our results demonstrate that Alz-50 rec- identity of the proteins that are recognized T cells (1). The two major subgroups that have structural proteins of HTLV, notably p24 bands of similar intensity. With normal
ognizes a protein present in neuronal ter- by Alz-50 remains to be elucidated. been characterized (6) are HTLV-I, which is and p19 (15), are not detectable in most AIDS hlIman serum, none of the antigens was
causatively linked to certain adult T-cell ma- patients (16). Instead, it seemed likely that detected (not shown). These results show
lignancies (7), and HTLV-II, which was first another member of the HTLV family might clearly that the antigens detected after virus
identified in a patient with hairy cell leuke- be involved in the etiology of AIDS. Here we infection are either virus-coded proteins or
mia (8). describe our studies of a group of cytopathic cellular antigens specifically induced by the
Viruses of the HTLV family have been yiruses (collectively designated HTLV-III) infection.
detected in some patients with the acquired isolated from patients with AIDS or pre- The antigens of H4/HTLV-IIl were also
immunodeficiency syndrome (AIDS) (9) AIDS. Isolation of these viruses was achieved compared with antigens from virus purified
or with pre-AIDS, a condition frequently by means of a novel system permitting the from the culture fluids of H4/ HTLV-III (Fig.
progressing to AIDS (10). A high proportion continuous growth of T-cell clones infected 2B). Extensive accumulation of p24 and p41
of patients with AIDS or pre-AIDS, as well as with the cytopathic types pf HTLV found in [see (20») occurred in the virus preparation
a significant number of hemophiliacs, have these disorders (17). We show that antigens (Fig. 2B, panels I and II). Protein stains
antibodies in their serum that recognize associated with human cells infected by showed that these molecules are the major
a cell surface glycoprotein (gp6l) that is HTLV-IlI are specifically recognized by components of the virus preparation (19).
present on certain human T cells infected antibodies in serum from AIDS and pre- P24 and p41 may therefore be considered
with HTLV-I (11). Gp6l and p65, a slightly AIDS patients, and present a preliminary viral structural proteins. Furthermore,
larger protein that is a homolog of gp6l and biochemical and immunological analysis of an antigen with a molecular weight of
occurs in another cell line producing HTLV-I, these antigens. approximately 110,000 was detected in the
were subsequently shown to be related to Lysates of two immortalized and infected virus preparation but was below limit of
the HTLV viral glycoprotein (12, 13). Studies human T-cell clones, H4/HTLVIII and H17/ detection in the cells. Also, p39 [see (20)]
HTLV-III (17), were tested with samples of was present iIi the virus preparation. It is
Laboratory of Tumor Cell Biology, National Cancer Institute, human serum in a strip radioimmunoassay interesting that p24 in the virus preparation
Bethesda, Maryland 20205. Program Resources, Inc. (RIA) based on the Western blot techniq1ue consistently appeared as a doublet (p24/
NCI-Frederick Cancer Research Facility, Frederick, Maryland 21701
(18). The sera were from patients with p23), whereas in the cells it appeared as
Department of Cell Biology, Litton Bionetics, Inc.,
Kensington; Maryland 20895. Laboratory of Tumor Cell Biology, AIDS or pre- AIDS, from contacts of such p24 alone. The significance of this is under
National Cancer Institute, Bethesda, Maryland 20205 patients, and from homo- or heterosexual investigation. P55 was not detected in the

24 sciencemag.org SCIENCE SCIENCE sciencemag.org Originally published 4 May 1984 in SCIENCE 25


W E S T E RN BLOT T ING : A G U I D E TO CU R R E N T M E TH O D S S E C T I O N T H R E E | RESEARCH ARTICLES

virus; however, the intensity of the p55 band and nucleic acid data clearly indicate that 15. V. S. Kalyanaraman, M. G. Sarngadlmran, P.
in the cells (Fig. 2B, lanes a) appeared to HTLV-ITJ is a true member of the HTLV A. BUdd, J. D. Minna, R. C. Gallo, Nature
correlate with the intensity of p24/p23 in family and that it is more closely related to (London) 294, 271 (1981); V. S. Kalyanaraman,
the virus preparation (Fig. 2B, lanes b), thus HTLV-II than to HTLV-I. M. Jarvis-Morar, M. G. Sarngadharan, R. C.
again suggesting a relation between these Gallo, Virology 132, 61 (1984).
antigens. The p55 is probably a precursor of REFERENCES AND NOTES 16. M. Robert-Gurotr et al.; in Cancer Cells, vol. 3,
p24, since a group-specific antigen of similar 1. M. Popovic, P. S. Sarin. M. Roben-Guroff, V. Human T·Cell Leukemia Viruses, R. C. Gallo
size (Pr 54gag ) in HTLV-I-infect~ ed cells is S. Kalyanaraman, D. Mann, J. Minowada, R. C. and M. Essex, Eds. (Cold Spring Harbor
the precursor of p24 and the other gag-coded Gallo, Science 219, 856 (1983); P. D. Markham, Laboratory, Cold Sprmg Harbor, N.Y., in press),
proteins (21). Occasionally an additional S. Z. Salahuddin, V. S. KalYanaraman, M. 17. M. Popovic, M. G, Sarngadharan. E. Read, R.
set of antigens was recognized by a serum Popovic, P. Sarin, R. C. Gallo, Jnl. 1. Cancer 31, C. Gallo, Science 224, 497 (1984).
(Fig. 2B, panel III) but their relation to the 413 (1983); S. Z. Salahuddin. P. D. Markham, F. 18. H. Towbin, T. Staehelin, J. Gordon, Proc. Natl.
antigens described above is unclear. Wong·Staal, G. FranChini, V. S. Kalyanaraman, Acad. Sci. U.S.A. 76, 4350 (1979).
Thus we have shown that viral or virus- R. C. Gallo, Virology 129, 51 (1983). 19. M. G. Sarngadharan, M. Popovic, L. Bruch, J.
Fig. 1. Serologic detection of antigens in HTLV-III producer cell Fig. 2. (A) Specificity of the antigens recognized. Lysates of
induced antigens in cloned human T cells 2. B. J. Poiesz, F. W. Ruscetti. A. F. Gazdar, P. Schupbach, R. C. Gallo, Science 224, 506
clones. Strip RIA were performed with human serum as described cloned cells before and after infection with HTLV-III were
infected with HTLV-ITJ are specifically A. Bunn, J. D. Minna. R. C. Gallo, Proc. Natl. (1984).
elsewhere in detail (21). Briefly, lysates of HTLV-IlI producer cell analyzed by the Western blot technique (18) with a I: 500
recognized by antibodies in the serum of Acad. Sci. U.S.A. 77, 7415 (1980). 20. Although in Fig, 2B the p41 in the virus
clones were subjected to electrophoresis under reducing conditions dilution of the serum shown in Fig. IB, lane e. (Lane a) The
patients with AIDS or preAIDS. The detection 3. H. M. Rho, B. Poiesz, F. W. Ruscetti, R. C. preparation appears to be larger than the p41
on preparative sodium dodecyl sulfate (SDS}-polyacrylamide slab HI7 clone before and (1ane b) the same clone after infection (HI7/
of p65 by many of the serum samples is of Gallo, Virology 112, 355 (1981); M. Seiki, S. in cells, the two molecules are of the same
gels, and electroblotted to nitrocellulose sheets (18). The sheets were HTLV-IlI); (lane c) the H4 clone before and (lane d) the same
special iriterest. We have tested these sera Hattori, Y. Hirayama, M. Yoshida, Proc. Natl. size. During application of the lysates to the
cut into strips. These were incubated with human serum diluted I: 100. clone after infection (H4/HTLV-III). All reactive antigens are
on strips prepared from Iysates of cells Acad. Sci. U.S.A. 80, 3618 (1983). gel in another experiment, a small amount of
After three thorough washings, bound antibodies of immunoglobulin virus-related with the exception of that with a molecular weight of
producing HTLV-I or -II. Some of these cells 4. M. Popovic et al.. in preparation. cellular lysate was spilled into the neighboring
0 (lgO) and immunoglobulin M (lgM) classes were made visible with 80,000 in HI7 cells; this antigen binds antibodies from all human
produce a p65 that has been shown (13) to 5. H. Mitsuya, H. G. Guo, M. Megson, C. O. lanes and the cellular p41 moved with the
radiolabeled, affinity-purified goat antiserum to human IgO and IgM sera investigated. Normal human serum did not bind to any of the
be coded for by the env gene of HTLV-I and Trainor, M. S. Reitz, S. Broder, Science 223, same velocity as the viral p41. A connecting
(H-chain specific) and autoradiography. (A) Analysis with H4/HTLV- virus-related bands (not shown). (B) Comparison of antigens in
to be the homolog of the gp61 described by 1293 (1984). band was thus formed between the p41 in
III cells. (Lanes a, d, and g) U.S. patients with AIDS; (lane b) a French (lanes a) cells and (lanes b) virus. Lysates ofH4/HTLV-III (250, µg
others (11, 12). Many of the sera recognizing 6. For a brief review, see M. G. Sarngadharan et the lane containing the cells and the p41 in
heterosexual male who developed AIDS after receiving a blood per lane) or virus purified from the cell culture ftuids (19) (51LS
p65 in HTLV-III-infected cells also al., in Human Carcinogenesi~.C. C. Harris and the lane with the virus. The same situation
transfusion in Haiti (24); (lane c) an AIDS patient from Switzerland; per lane) were analyzed with 1:500 dilutions of human sera. (Panel
recognized, though somewhat faintly, p65 H. H. Autrup, Eds. (Academic Press, New occurred with p39 in cells and virus.
(lane e) a normal heterosexual control; (lane 0 a French pre-AIDS I) Same serum as in Fig. 2A; (panel II) serum ofa’Swiss male
in cells producing HTLV-lor -II, and some of York, 1983), p. 679. 21. J. Schupbach, V. S. Kalyanaraman, M. G.
patient (24); (lane h) a Swiss heterosexual drug addict; (lane i) a homosexual with fatigue and generalized lymphadenopathy (pre-
them also recognized gag-related antigens 7. V. S. Kalyanaraman, M. G. Sarngadharan. Y. Sarngadharan, R. C. Gallo, in preparation.
normal homosexual control. (B) Analysis with H17/HTLV-IlI cells. AIDS); (panel III) serum from same AIDS patient as in Fig. IB, lane
(data not shown). Nakao, Y. Ito. T. Aoki, R. C. Gallo, Proc. Natl. 22. M. G. Sarngadharan et al., in preparation.
(Lane a) An infant with AIDS whose mother is a prostitute; sera from d. An antigen with a molecular weight of 110,000 and p41, p39, and
In addition, the reaction of some human Acad. Sci. U.S.A. 79, 1.653 (1982); M. Robert” 23. S. Arya et al., in preparation.
both are highly positive for antibodies to the HTLV membrane antigen p24 are enriched in the virus preparation [see (20)]. The serum
sera with virus-related antigens of HTLV-ITJ- Guroff, Y. Nakao, K. Notake, Y. Ito, A. H. 24. J. B. Brunet et al., Lancet 1983-1,700 (1983).
(11, 25) and in our ELISA with disrupted HTLV-III (19); (lane b) same in panel III recognized a subset of the antigens recognized by the
infected cells could be partially inhibited by Sliski, R. C. Gallo, Science 215, 925 (1982); Y. 25. M. Essex, personal communication.
serum as in (A), lane d; (lane c) normal heterosexual control; (lane sera used in panels I and II.
large amounts of extracts of cells producing Hinuma et al., Int. J. Cancer 29, 631 (1982); 26. M. Popovic, V. S. Kalyanaraman, D. L. Mann,
d) another Swiss AIDS patient; (lane e) a Swiss heterosexual male
HTLV-II (Fig. 3). When a human serum not W. A. Blattner et al., ibid. 30, 257 (1982); E. Richardson, P. S. Sarin, R. C. Gallo, in
intravenous drug abuser with generalized lymphadenopathy and
recognizing p65 was used, the antigens for J.Schupbach, V. S. Kalyanaraman, M. G. Cancer Cells, vol. 3, Human T-Cell Leukemia
thrombocytopenic purpura (pre-AIDS).
which there was competition included p55, Sarngadharan, Y. Nakao, R. C. Gallo, ibid. 32; Viruses, R. C. Gallo and M. Essex, Eds. (Cold
p48, p41, p39, and p24. These results were 583 (1983); J. Schupbach, V. S. Kalyanaraman, Spring Harbor Laboratory, Cold Spring Harbor,
confirmed by the demonstration that a rabbit M. G. Sarngadharan, W. A. Blattner, R. C. Gallo, N.Y., in press).
antiserum raised against purified HTLV-III Cancer Res. 43, 886 (1983). 27. R. C. Gallo et al., Science 224. 500 (1984).
Fig. 3. Relation between HTLV-
showed some reactivity with antigens of 8. V. S. Kalyanaraman et aI., Science 218, 571
III and HTLV-II. Serum of an
HTLV-II and, to a lesser extent, with HTLV-I. (1982); E. P. Gelmann et al., Proc. Natl. Acad. ACKNOWLEDGEMENTS
AIDS patient at a dilution of I:
In contrast, antiserum to HTLV-II recognized Sci. U.S.A. 81, 993 (1984). We thank J. Ahmad for technical assistance and
500 was tested in a competition
both HTLV-I and -III antigens, and an 9. R. C. Gallo et al., Science 220, 865 (1983); E. R. Luthy and M. Vogt, Division of Infectious
RIA on strips (20) prepared with
antiserum to HTLV-I reacted well with P. Gelmann et al., ibid., p. 862. Diseases, Department of Medicine, University
H4/HTLVIII cells. (Lane a) The
HTLV-Ii, but only faintly with HTLVITJ (22). 10. F. Barre-Sinoussi et al., ibid., p. 868. Hospital, Zurich, and O. Haller, Institute for
human serum was added directly
Moreover, nucleotide sequences of HTLV- 11. M. Essex et al., ibid., p. 859; M. Essex et al., Immunology and Virology, University of Zu·
to the strip (uncompeted control);
ITJ have been found to be related to HTLV-I ibid. 221, 1061 (1983). rich, Zurich, Switzerland, for making some sera
(lanes b to e) the serum was first
and -II (23). Although the morphology of 12. T. H. Lee, J. E. Coligan, T. Homma, M. F. from AIDS and pre-AIDS patients available and
absorbed for 3 hours at 37”C with
HTLV-ITJ particles appears to be somewhat McLane, N. Tachibana, M. Essex, Proc. Natl. for providing clinical information. J .S. is a Fogarty
1 mg of cellular extract. In (b) the
different from the morphology of HTLV-I and Acad. Sci. U.S.A., in press. International Fellow of the National Cancer
absorption was with uninfected
-II (Fig. 4), and although some differences are 13. J. Schupbach, M. G. Sarngadlmran, R. C. Gallo, Institute.
H4 cells (not producing virus); in
also found in the protein patterns of purified Science, in press.
(c) the absorption was with H4/
virus preparations (19), these immunological 14. H. W. Jaffe et al., Science 223, 1309 (1984). 30 March 1984; accepted 19 April 1984 505
HTLV-IlI cells producing HTLV-
III (positive control); in (d) the
absorption was with C3/44 cells
(26) producing HTLV-II; in (e) the
absorption was with HUT 102
cells producing HTLV-I (2).
Fig. 4. Electron microscopy of thin sections of cells producing HTLV-I, -II, and
-III. (Top) HUT 102 cells producing HTLV-I (2). (Middle) Cells from an AIDS patient
(J.P.) producing HTLV-II (24). (Bottom) Cells from a patient [described in (27)]
with pre-AIDS, producing HTLV-III. (Panels a) Virus particles budding from the cell
membrane. (Panels b) Free particles have separated from the membrane. (Panels c)
Free particles sectioned in a different plane, Note the dense, cylindrical core region
of HTLV-II1.

26 sciencemag.org SCIENCE SCIENCE sciencemag.org 27


W E S T E RN BLOT T ING : A G U I D E TO CU R R E N T M E TH O D S S E C T I O N T H R E E | RESEARCH ARTICLES

Cell surface P-glycoprotein associated Each of the different mammalian cell lines
examined in this study (Table 1) was originally
Fig. 1. Electrophoretic and immunochemical
analysis of plasma membrane components.

with multidrug resistance in


selected for resistance to a specific drug, and Cell culture, plasma membrane isolation,
in each case a multidrug resistance pheno- and electrophoresis were carried out as de-
type typified by cross resistance to unrelated scribed previously (21, 22, 27). (A) Gels were

mammalian cell lines compounds was observed. Such a phenotype


appears to reflect a membrane-associated al-
loaded with 10 µg of membrane protein per lane.
They were overstained by the silver staining
teration (6, 24, 26). We therefore pre- pared method of Switzer et al. (34) and were then re-
Norbert Kartner, John R. Riordan, and Victor Ling membranes from these cell lines component duced to acceptable intensity by soaking in a so-
could not be detected by for analysis of their lution of 0.03M ferric ammonium sulfate in 0. 18M
The plasma membranes of hamster, mouse, and human tumor cell lines that display components by sodium dodecyl sulfate-poly- sulfuric acid diluted 1:10, followed by a final rinse
multiple resistance to drugs were examined by gel electrophoresis and immunoblotting. acrylamide gel electrophoresis (SDS-PAGE) in 0.3M sodium carbonate and thorough wash-
In every case, increased expression of a 170,000-dalton surface antigen was found to be (22). For CHR CHO cells (lanes b to d in Fig. ing in water. Molecular size standards (Bio-Rad)
correlated with multidrug resistance. This membrane component is of identical molecu- IA), a protein having a molecular size of about are shown in the first lane. These consist of 0.1
lar size and shares some immunogenic homology with the previously characterized 170,000 daltons appeared in the plasma mem- µg each of myosin, (200,000), β-galactosidase
P-glycoprotein of colchicine-resistant Chinese hamster ovary cells. This finding may have brane with a staining intensity proportional (116,250), phosphorylase b (92,500), bovine
application to cancer therapy. to the degree of resistance to the drug. As serum albumin (66,200), ovalbumin (45,500),
previously characterized, this band is referred carbonic anhydrase (31,000), soybean tryp-
to as the Pglycoprotein (20, 22). Similarly sin inhibitor (21,500), and lysozyme (14,400).

S
election of variants in mammalian cells cellular accumulation of the drugs involved stained bands of approximately the same mo- (Lane a) Drug-sensitive parent CHO cell, AUXB
that are resistant to specific drugs, such (5-19), and changes in the plasma membrane lecular size are seen in the other drugresistant I; (lane b) colchicine (0.10 µg/ml)-resistant CHO
as Vinca alkaloids, maytansine, colchi- have been observed (17- 23). In the well-char- cell lines that we examined (lanes e, h, j, and cell CHRA3; (lane c) colchicine (3.0 µg/ml)- re-
cine, anthracyclines, actinomycin D, acterized colchicineresistant (CHR) Chinese 1 in Fig. IA). Such a protein staining in the sistant CHO cell CHRB3; (lane d) colchicine
or bleomycin, is often accompanied by hamster ovary (CHO) system, for example, corresponding drug-sensitive parental cells (10 p.g/ml)-resistant CHO cell CHRC5; (lane e)
expression of a complex phenotype of cross genetic analyses involving cell-cell hybrids, (lanes a, g, i, and k) or drug-sensitive rever- daunorubicin-resistant CHO cell DNRR51; (lane
resistance to various unrelated drugs (1-14). drug-sensitive revertants, and DNA-medi- tant (lane f ). f) colchicine-revertant CHO cell 110 (derived
This characteristic is referred to as the mul- ated transformants of the CHR phenogenesis As a means of further characterizing the from CHRC5); (lane g) sensitive parent SV40-
tidrug resistance phenotype. The generation type indicate that multidrug resistance, col- relatedness of the 170,000-dalton membrane transformed Syrian hamster cell Cl2TSV5S; (lane
of such variants in tumor cells may be an im- chicine resistance, and reduced drug accu- components observed in these drug-resistant h) actinomycin D-resistant SV40-transformed
portant mechanism by which neoplasms be- mulation are the result of the same genetic lines, an antiserum to plasma membrane Syrian hamster cell Cl2TSV5R2; (lane i) sensitive
come resistant to treatment by combination alteration (21, 24, 25). Moreover, the expres- vesicles of the highly colchicine-resistant mouse L cell LMTK-; (lane j) colchicine (0.50 µg/
chemotherapy. sion of a 170,000-dalton plasma membrane CHO line CHRC5 was prepared. The specificity ml)-resistant mouse L cell ECHR; (lane k) sensi-
Studies in model systems indicate that glycoprotein (P-glycoprotein) is invariably of this antiserum for the P-glycoprotein tive parent human lymphoid cell CCRF-CEM;
multidrug resistance results from a reduced associated with this pleiotropic phenotype was improved by cross absorption with (lane 1) vinblastine-resistant human lymphoid
(20-22, 24). The degree of drug resistance is immobilized, detergent-solubilized plasma cell CEM/VLB100 (see Table I for further de-
Ontario Cancer Institute, Princess Margaret Hospital, and correlated approximately with the amount of membrane proteins of the parental drug- tails). The P-glycoprotein region is indicated (P)
Department of Medical Biophysics, University of Toronto, Toronto, Pglycoprotein present (20, 22). The objective sensitive line (21, 27). The absorbed antiserum at molecular size 150,000 to 170,000 daltons.
Ontario, M4X IK9, Canada. Research Institute, Hospital for of the present study is to determine whether was then used to examine the membrane Lanes a to f and g to I represent two separate
Sick Children, and Departments of Biochemistry and Clinical
or not P-glycoprotein expression is also asso- components of the lines shown in Fig. lA. gels that were run simultaneously and treated
Biochemistry, University of Toronto, Toronto, Ontario, MSG
IX8. Ontario Cancer Institute, Princess Margaret Hospital, and ciated with the multidrug resistance pheno- Each of the drugresistant lines expressed a identically. (B) SDS-PAGE was performed as in
Department of Medical Biophysics, University of Toronto type observed in other cell systems. 170,000-dalton component that was stained (A), with the exception that 50 µg of membrane
protein was loaded per lane. Western blots were
overlaid with previously absorbed rabbit anti-
Table 1. Description of cell lines. Cell line CHRA3 was selected from AUXB1; CHRB3 was selected from CHRA3 in a second step; and CHRC5 was serum (diluted 1:100) against isolated plasma
selected from CHRB3 in a third step (6). DNRR51 was selected from AUXBI in two steps (27). 110 is a revertant cell line, selected in a single step from membranes from the colchicine-resistant CHO
CHRC5 (24). Abbreviations for drugs are: ACR, acriflavine; ADR, adriamycin; AMD, actinomycin D; CCH, colchicine; CMD, colcemid; CYB, cytochalasin B; cells CHRC5 and processed as described (21,
DNR, daunorubicin; EME, emetine; ERY erythromycin; ETB, ethidium bromide; GRD, gramicidin; PRO, proflavin; PUR, puromycin; VCR, vincristine; and VLB, 27). Lanes a to f and g to I are two separate blots
vinblastine. Relative resistance was calculated as the ratio of drug concentration tolerated by the resistant cells to that tolerated equally by the sensitive prepared and treated identically, but lanes k and
parent cell. Tolerance was assessed by relative growth rates except for cell lines 110 and ECHR, whose colony-forming abilities in the presence of drug were I were exposed to film four times longer than
compared with the appropriate parental lines. Since cross resistance is shown only for drugs that have been tested and the results reported, absence of other lanes. All lanes are as described in (A). (C)
a drug from the column on cross resistance does not imply lack of cross resistance to that drug. The drugs are listed in order of decreasing relative cross SDS-PAGE was performed as in (B), but blots
resistance. were overlaid with unabsorbed serum prepared
against CEM/VLB100 plasma membranes (di-
luted 1:100). Lanes k and I were exposed to film
for one-tenth the exposure time of other lanes.

by this antiserum (Fig. 1B). It is clear from the of the presumptive human P-glycoprotein proteins from drug-sensitive cells, and many
size and cross-reactivity of this component by the antiserum is likely due to reduced antigens common to both drug-resistant and
that it is similar to the P-glycoprotein of the cross-reactivity of this component with the drug-sensitive human cells were strongly
CHR CHO cells. Staining of other components antiserum against the CHO cell P-glycoprotein. stained (lanes k and 1; note the exposure
of about 50,000 and 200,000 daltons is also To further corroborate the above conclu- time). This crude antiserum stained only the
observed with this antiserum. We believe sions, we stained the membrane components drug resistance-associated P-glycoprotein in
that these components are not related to with an antiserum to plasma membrane of the rodent cell membranes (Fig. IC). Thus, of
the P-glycoprotein or multidrug resistance the vinblastine-resistant, human cell line, the dozen or so different cell surface antigens
because they vary with different preparations CEM/VLB100 (Fig. IC). In this case, the serum that can be detected with the two antiserums,
of membranes. The relatively faint staining was not previously absorbed with membrane only the P-glycoprotein is consistently stained

28 Originally published 23 September 1983 in SCIENCE SCIENCE sciencemag.org 29


W E S T E RN BLOT T ING : A G U I D E TO CU R R E N T M E TH O D S

in all drug-resistant mammalian cell lines species of drug-resistant cells established in 19. W. T. Beck and M. C. Cirtain, Cancer Res. 42,
tested. These observations, and the fact that
a side-by-side comparison of the different P-
glycoprotein bands reveals no significant dif-
vitro. It is reasonable to suppose that such
a relationship might also exist in vivo. If P-
glycoprotein is commonly present in tumor
184 (1982).
20. R. L. Juliano and V. Ling, Biochim. Biophys.
Acta 455, 152 (1976).
GE Technotes
ference in molecular size, as shown in Fig. 1, cells from patient biopsies, and if this antigen 21. P. G. Debenham, N. Kartner, L. Siminovitch, J.
strongly indicate that the P-glycoprotein is is expressed in increased amounts in tumors R. Riordan, V. Ling, Mol. Cell. Biol. 2, 881
conserved relative to other mammalian mem- nonresponsive to treatment by combination (1982).
brane antigens that are detectable by Western chemotherapy, immunochemical screening 22. J. R. Riordan and V. Ling, J. Biol. Chem. 254,
blotting. for the antigen could provide a rapid diag- 12701 (1979).
Our observations that P-glycoprotein is nostic basis for planning treatment of cancer 23. R. H. F. Petersor and J. L. Biedler, J. Supramol.
present in increased amounts in various patients. Moreover, hitherto unresponsive Struct. 9, 289 (1978).
drug-resistant lines, and that in the CHR CHO tumors may become amenable to treatment 24. V. Ling, Can. J. Genet. Cytol. 17, 503 (1975).
system the amount of Pglycoprotein expressed with Pglycoprotein- targeted antibodies con- 25. __ and R. M. Baker, Somat. Cell Genet. 4,
is correlated with the degree of resistance, are jugated with toxins. 193 (1978).
consistent with a mechanism of resistance 26. R. M. Baker and V. Ling, in Methods in
involving gene amplification. The appearance REFERENCES AND NOTES Membrane Biology., E. D. Korn, Ed. (Plenum,
of double minute chromosomes has been 1. N. T. Bech-Hansen, J. E. Till, V. Ling, J. Cell. New York, 1978), vol. 9, p. 337.
correlated with unstable drug resistance Physiol. 88, 23 (1976). 27. N. Kartner, M. Shales, J. R. Riordan, V. Ling,
in known gene-amplified systems (28, 29). 2. S. Brabbs and J. R. Warr, Genet. Res. 34, 269 Cancer Res., in press.
Double minute chromosomes have also (1979). 28. P. C. Brown, S. M. Beverley, R. T. Schimke,
been observed in association with multidrug 3. L. J. Wilkoff and E. A. Dulmadge, J. Natl. Mol. Cell. Biol. 1, 1077 (1981).
resistance in several mouse cell lines Cancer Inst. 61, 1521 (1978). 29. R. J. Kaufman, P. C. Brown, R. T. Schimke,
including colchicine- resistant lines for which 4. B. Salles, J.-Y. Charcosset, A. Jacquemin-Sab- ibid., p. 1084.
the degree of resistance and P-glycoprotein Ion, Cancer Treat. Rep. 66, 327 (1982). 30. S. M. Robertson, V. Ling, C. P. Stanners, in
expression correlate with the number of 5. T. Skovsgaard, Cancer Res. 38, 4722 (1978). preparation.
double minutes contained within the cells (14, 6. V. Ling and L. H. Thompson, J. Cell. Physiol. 31. B. P. Kopnin, Cytogenet. Cell Genet. 30, 11
30). Double minutes have also been reported 83, 103 (1974). (1981).
in multidrug-resistant hamster cells that were 7. K. Dan0, Acta Pathol. Microbiol. Scand. Suppl. 32. We have recently observed, using the
originally selected for colchicine resistance 256, 11 (1978). methods
(30, 31). This speculation on the origin of the 8. M. P. Chitnis and R. K. Johnson, J. Natl. described, highly elevated P-glycoprotein
P-glycoprotein in drug-resistant cells suggests Cancer Inst. 60, 1049 (1978). expression in vincristine-resistant Chinese
its preexistence, in much smaller amounts, in 9. J. L. Biedler and R. H. F. Peterson, in Molecular hamster cells, which bear a chromosomal
the drug-sensitive parent cell. In this context, Action and Targets for Cancer Chemothera- homogeneous staining region [see T. Kuo et
we observed a barely detectable, antigenically peutic Agents, A. C. Sartorelli et al., Eds. al. in Gene Amplification, R. T. Schimke, Ed.
cross-reactive band of the same molecular (Academic Press, New York, 1981), p. 453. (Cold Spring Harbor Laboratory, Cold Spring
size as the P-glycoprotein in all the drug- 10. Y. Langelier, R. Simard, C. Brailovsky, Harbor, N.Y., 1982), pp. 53-57].
sensitive cells examined in Fig. lB when the Differentiation 2, 261 (1974). 33. N. T. Bech-Hansen et al., J. Natl. Cancer Inst.
film was exposed for a much longer period 11. P. D. Minor and D. H. Roscoe, J. Cell Sci. 17, 59, 21 (1977).
(data not shown). What specific role the 381 (1975). 34. R. C. Switzer, C. R. Merril, S. Shifrin, Anal.
Pglycoprotein might play in the maintenance 12. V. Crichley, D. Mager, A. Bernstein, J. Cell. Biochem. 98, 231 (1979).
of the multidrug resistance phenotype, or in Physiol. 102, 63 (1980).
the drug-sensitive cells, is unknown. It is also 13. C. D. Aldrich, J. Natl. Cancer Inst. 63, 751 ACKNOWLEDGEMENTS
not yet known whether the P-glycoprotein is (1979). Supported by grants from the National Cancer
expressed in normal tissues. 14. F. Baskin, R. N. Rosenberg, V. Dev. Proc. Institute of Canada and the Medical Research
Our findings could have important implica- Natl. Acad. Sci. U.S.A. 78, 3654 (1981). Council of Canada. We thank W. T. Beck, P. G.
tions for cancer therapy. It is possible that clin- 15. S. A. Carlsen, J. E. Till, V. Ling, Biochim. Debenham, T. Kuo, and R. Simard for making
ical resistance to combination chemotherapy Biophys. Acta 455, 900 (1976). available their drug-resistant cell lines; and N.
might result from an unchecked proliferation 16. M. E. Lalande, V. Ling, R. G. Miller, Proc. Alon, S. Fahim, and M Naik for technical
of tumor cell subpopulations with a multi- Natl. Acad. Sci. U.S.A. 78, 363 (1981). assistance.
drug resistance phenotype (33). The present 17. D. Kessel and H. B. Bosmann, Cancer Res. 30,
data indicate that there is a strong correlation 2695 (1970). 13 May 1983
between the expression of multidrug resis- 18. D. Garman and M. S. Center, Biochem.
tance and surface P-glycoprotein in different Biophys. Res. Commun. 105, 157 (1982).

30 sciencemag.org SCIENCE
GE Technotes

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© 2015 General Electric Company—All rights reserved. First published May 2015.
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