ELECTROPHORESIS

Prepared by-
Unnati sahu
Msc(bio-Chemistry)
 Synopsis
• Introduction
• History
• Principle
• Electrophorelic mobility
• Types of electrophoresis
• Paper electrophoresis
• Starch gel electrophoresis
• Agarose gel electrophoresis
• references
 Introduction of
electrophoresis
• Electrophoresis is the Migration of charged particles or
molecules in a medium under the influence of applied electric
field.
• Father of electrophoresis Arne Tiselius( 1902-1971)
• Electro -Electric Phoresis-Migration carry
• This separation technique based on the different migration
features of charged
molecules in a electric field.
 History

• A russian physicist alexander reuss madethe first

electropholetic observation of colloidal paxticles in 1807.
• Arne tiselius – father of electrophoresis
 Principle
• Electrophoresis is a method used to separate charged particles
from one another based on difference in their migration speed
• It is depending upon two electrodes
• Is depending upon two electrodes and they are Immersed in
two separate buffer chamber
• The particle can migrate from one chamber to the another by
using an electric power supply.
• Positive lon (cations) move towards the negative electrode.
• Negative ion(anions) move towards positive electrode.
 Contd...
• Electrons flow in the external current from the positive
electrode to the negative electrode
• Any charged ions or molecules migrates when placed in an
electric field.
• The rate of migration depending upon its size shape and
applied electric current
• V = E *q/F
• V = velocity of Migration of the molecules
E = electric field in volts per centimeter
q = net electric charge on the molecule
F = Frictional coefficient
 B-1 electrophoretic mobility
• lon placed in an electrical field (E) experiences force Fef that is
proportional to field strength and the charge (q) on the ion (Fef
=q.E)
• As the ion moves, a frictional force (ffr) opposes the forward
movement of the ion
• In a constant electrical field the velocity of particle is constant
and depends on the balance between the two forces
electrophoretic mobility is the fundamental parameter which
determines the efficiency of separation based on charge to size
ratio (g)
• Change in pH effectively alters the charge on the ions and their
electrophoretic mobility.
 Types of electrophoresis
• The two main electrophotic methods are:-
1) Moving boundary electrophoresis :- in which saparation is
carried out in the absence of a supporting meadium.
2) Zone electrophoresis :- in which paper of gel is used as
supporting medium for separation .here the charged particles
are separated onto different zones or bands.This is of two
types as
i) paper electrophoresis
ii) gel electrophoresis
 Paper electrophoresis
• The technique of paper electrophoresis is simple and
inexpensive and requires only micro quantities of plasma
for separation.
• The support medium is a filter paper
• The electrophoresis apparatus in its simplest form consists of
two troughs to contain buffer solution, through which electric
current is passed.
• Frequently used in isolating proteins, amino acids and
oligopeptides.
 Principle of paper
electrophoresis

• The charge carried by a molecule dependson the pH of the

medium . Electrophresis at low voltage on not usually to
seprate low molecular waight compound because of diffusion
but it is easier to illustrate the relationship between charge and
pH with amino acids than proteins (or) other macromolecules.
 Apparatus
• 1. Power pack: DC o-500 V or o-150mA
• 2. Electrophoretic Cell: electrodes, Buffer reservoirs,
transparent insulating cover etc.
• Sample application: Spot/streak
• Electrophoretic run: Sample equilibrated with buffer
• Over heating avoided by cold room
• < 2hrs
• Paper dried @1o°C
• Detection: by comparing with standards
• 1. Fluorescence 2. UV-absorption (260-28onm) 3. Staining
• Clinical application: Serum proteins etc
 Procedure
• A long strip of filter paper is moistened with a suitable
buffer solution of the desired p H and the sample is applied
transversely across the central part of the strip.
• Ends are fixed to dip in buffer solutions in two troughs
fitted with electrodes
• Electric field of about 20 volts/cm is established.
• The charged particles of sample migrate along the strip
towards respective electrodes of opposite polarity,
according to net charges, sizes and interactions with the
solid matrix
Contd...
• Homogeneous group of particles migrate as a separate
band
• The electrophoresis is carried out for 16-18 hours.
• Separated Proteins are fixed to a solid support using a
fixative such as Acetone or Methanol
• Proteins are stained (bromophenol blue) to make
them visible
• The separated proteins appear as distinct bands
• Drawback-long time interval and blurring of margins
 Observation

• The different fractions appear as blue coloured bands across

the filter paper starting from the moving boundary backwards
• If a quantitative estimation is required for each fraction, the
bands may be carefully cut and eluted or the bands may be
scanned optically in a densitometer
• In human plasma five different bands can be identified on
paper electrophoresis
 Starch gel electrophoresis
• Introduction:- starch gel as a stabilizing medium for zone
electrophoresis was introduced by smithies in 1955.
• 2 forms :- a amylose and amylopectin branched polymers.
• mostly used for protein separation
 Apparatus
• The starch is Apparatus consists of two buffer Chambers. Each
compartmentalized and connected by paper wicks. the
electrode is kept in one chamber and the other chamber is
connected to the gel tray.
• starch is hydrolysed in acidified acetone at 37 °Ⅽ the
suspension is then neutralized with Sodium Acetate and West
with a large amount of distilled water and acetone.
• this hydrolyzed starch when heated and cooled in an
appropriate buffer set as a gel
 Procedure
• The sample is applied to the gel by soaking a small piece of
filter paper and inserting it in the gel
• The main drawback of starch gel is its unlimited pore size
• Moreover it is difficult to prevent contamination of starch gel
by microorganisms
• Another disadvantage of the starch is that upon stemming to
detect the separated components the stars gel tones effective
making direct photoelectric determination impossible
• However the resolving power of starch is very high
• Starch gel does not absorb protein hence the separation of
proteins gives sharp bends without teiling
 Contd...
• the gel can be made transparent by soaking in glycerol heated
to 70-80 °C for 5 minutes
• Then the gel can be densitometrically scanned start gel has no
denaturing effect on enzymes
• To analyse the presence of active enzymes that specific
substrate solution is poured in drops on the gel
• The appearances of coloured band indicates the presence of
active enzymes the result can be immediately recorded by
photography by using appropriate filter
 Introduction Of Agarose Gel
Electrophoresis
• Agarose gel electrophoresis is a method to separate DNA or
RNA molecules by size.
• This is achieved by moving negatively charged nucleic acid
molecules through an agarose matrix with an electric field
(electrophoresis).
• Shorter molecules move faster and migrate
faster than longer ones
 Material required for agarose
gel electrophoresis
• Electrophoresis chamber
• Agarose gel
• Gel casting tray
• Buffer
• Staining agent (dye)
• A comb
• DNA ladder
• Sample to be separate
 Types of agarose
• Standard Agarose LE
Gels at 35-38°C; Melts at 90-95 C
Becomes opaque at high concentrations

• Low Melting Agarose (NuSieve)

Gels at 35°C; Melts at 65°C
Often used to isolate DNA fragments from gel
 Preparation of agarose gel
• In this electrophoresis technique agarose is used at a concentration
of 1 to 31.
• agarose gels are prepared by suspending day agarose in aqueous
buffer and then boiling the mixture until aqueous solution is formed
• It is allowed to cool to room temperature to form a rigid gel
• Numerous Inter and intermolecular hydrogen bonds within and
between the long agarose chain molecules are responsible for the
gelling properties
• This cross linking gives the gel good ant convectional properties
• The pore size is controlled by the initial concentration of agarose
• Low concentrations of agarose produce large pore size and high
concentrations produce smaller pore size
Agarose Gel Electrophoresis
 Procedure
• In agarose gel electrophoresis techanique the galss slides on
which agarose gels are prepared by kept on the bridges of the
apparatus.
• The gel is connected to the anode and cathode buffer
reservoirs throught paper wicks.
• The sample is applied in the wells made in the gel when
electrophoresis is complete the DNA is identified by
immersing the gel in a dillute solution of the flurescent dye
,elhidium bromide.
 Contd...
• After some time the gel is washed to remove unbound dye
from the agarose and then it is illuminated with ultraviolet
light to excite the fluorescence of ethidium bramide.

• The gel with its flourescent bands is then photographed

agarose gel can be used for immunoelectrophoresis
techanique.
 Application of electrophoresis
• Estimation of the DNA molecule.[ Agarose, PAGE]
• Analysis of PCR product.[ Agarose ]
• Separation of restricted genomic DNA and RNA.[Agarose
and PAGE respectively]
• Conformation of newly isolated DNA.[Agarose]
• Separation of most small fragments of DNA.[PAGE
• In forensic science.[Agarose, PAGE, SDS-PAGE, 2D PAGE
,Capillary gel electrophoresis, PFGE]
• In determining molecular wt. of protein.[SDS-PAGEL.ete
 References

• Keith wilson – principle and techniques of biochemistry and

molecular biology.
• Upandhyay – biphysical chemistry
• bioinstrumentation