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TEKNIK ISOLASI PROTEIN

Protein ekstraseluler Tahapan isolasi

Sentrifugasi cairan sel/ medium kultur menghasilkan : Supernatan (crude extract) Pelet (sel & komponen non-protein)

Protein intraseluler Tahapan isolasi :

Cuci jaringan/ sel dengan bufer salin : untuk menghilangkan pengotor/ bahan ekstraseluler Sel mikroba : sentrifugasi & resuspensi dalam bufer Lisis sel memecah/membuka sel ekstrak : homogenat Menggunakan mortar/pestle, homogenizer, sonicator, tissue grinder, cell disruptor, blender Menghasilkan : homogenat Sentrifugasi menghasilkan : pelet (mengandung protein membran) supernatan (crude extract)

Cell disruption methods


Cell lysis method Blade homogenization Hand homogenization Kind of tissue Most animal, plant tissue Soft animal tissue

Sonication
French pressure cell Grinding with alumina/ sand Glass bead vortexing Enzyme digestion Detergent lysis Organic solven lysis Osmotic shock

Cell suspention
Bacteria, yeast, plant cell Bacteria, plant cell Cell suspention Bacteria, yeast Tissue culture cell Bacteria, yeast, plant cell Erythrocytes, bacteria

Removal of Non-Protein Components

PROTEIN SEPARATION
The goal of a protein separation is to obtain the protein in a

pure, active form using a minimum number of steps The shortest time possible.

SEPARATION METHOD
Chromatographic Methods Proteins differ in size and charge Ion exchange chromatography Anion and Cation Gel Filtration (size exclusion) Separates based on size Affinity Methods Affinity for ligands Engineered affinity sites, e.g. histidine tag

Chromatography

Solubility techniques
Salting out methods Solubility sensitive to ionic strength. Initial salting in and thensalting out At high salt, solvent tied up with interacting with salts so that it is insufficient to solubilize proteins. 1st go to maximal salt that protein target is soluble, centrifuge and discard pellet. Now add just enough salt to bring down protein. Collect pellet. Organic solvents Same principle as salting out; taking advantage of different solubilities. Avoid totally denaturing proteins. pH Proteins have many ionizable groups with range of pKs. When net charge of protein is zero, this is the isoelectric point or pI. Proteins are typically least soluble at their pI due to minimizing charge charge interactions. Crystallization The solubility methods where proteins are ppt can be used to grow crystals of proteins. This is only done when the protein is relatively pure

Ammonium sulfate precipitation (salting out)


When high concentration of salt are present, proteins

tend to aggregate and precipitate out of solution : salting out Different proteins precipitate at different salt concentration. pH, temperature and protein purity play important roles in determining the salting out poin. Ammonium sulfate is the salt choice because it combines many useful feature :

salting out effectiveness pH versality high solubility low heat of solution low price

Ammonium sulfate concentration : % saturation Simple equation for calculation of gram ammonium sulfate needed to make an

X% solution from Xo% : 515 (X-Xo) g = ------------100-0,27X

Dialysis

ION EXCHANGE CHROMATOGRAPHY


Low salt P+ + Na+ High salt Na+ + P+

IONIC ELUTION pH ELUTION

Ion exchange groups used in protein purification

STRONG CATION SO3 Sulpho CH2SO3 Sulphomethyl C3H6SO3 Sulphopropyl WEAK CATION Carboxy Carboxymethyl

COO CH2COO-

STRONG ANION CH2N+(CH3)3 Triethylaminomethyl C2H4N+(C2H5)3 Triethylaminoethyl C2H4N+(C2H5)2CH2CH(OH)CH3 Diethyl-2-hydroxypropylaminoethyl WEAK ANION C2H4N+H3 Aminoethyl C2H4N+H(C2H5)2 Diethylaminoethyl

Gel Filtration
Porous beads made of different materials.

Size of pores can be controlled Small molecules small enough to go into beads whereas larger go around

and thus flow faster. There is exclusion limit (all proteins too large to go into pores).
Can be used as preparative method or be

used to determine molecular size Gels made of dextrans, agarose or polyacrylamide

Affinity Method
A ligand which has tight binding

to protein is attached to matrix.


Protein of interest binds but

others pass through Elute using soluble ligand. Beside small molecules, can also use antibodies Recent advances in molecular biology entails engineering a poly his group which binds to Ni or use parts of other proteins and columns that bind to this other protein

Addition of glucose (G)

Visualization &/or Isolation of a protein


proteins migrate in an electrical field at rates that depend

upon their net charge, size, and shape Gel Electrophoresis... separation by charge (separation of in a media as gels) in a media as porous gel (starch/polyacrylamide) : gels & staining Isoelectric Focusing*: proteins are separated in a gel of a continuous pH gradient, proteins move to point in gel equal to its pI, i.e., no charge SDS-PAGE*: Sodium Dodecyl Sulfate - polyacrylamide gel electrophoresis... proteins treated with ionic detergent that separates according to size SDS binds to protein @ 1 SDS/2 aa's thus proportional to a protein's MW

SDS-PAGE

Identification of proteins presence & & its quantification

Identification - is often done by spectrophotometry spectrophotometers measures intensity of light beam before & after light passes through a liquid solvent with sample dissolved in it, (in a cuvette)... compares the two light intensities over a range of wavelengths. Percent transmittance... ratio of intensity of light passing through the sample to the intensity of light shining on sample multiplied by 100%. Absorbance... is the log of the transmittance

instruments... Spectronic 20/ spectrophotometer UV/ Vis

SPECTROPHOTOMETRIC METHODS of DETECTING PRO TEINS


UV absorbance at 280 nm. (measures aromatic aa's) Colorimetry reactions - colored dye binds to amino acids Ninhydrin reaction - rx's w amino = blue color (10-9 M) Biuret test = mg quantities based on Copper ion binds stiochiometrically = violet color Bradford test = ug amounts based on dye Coomassie blue - binds to peptide Fluoroescamine dye = pg quantities... (10-12 M) Quantification of amounts of protein present Quantification is based on BEER-LAMBERT Law linear relationship between... light Absorbance vs. Conc Protein Standard Curve

Quantification of Protein Concentrations with ENZYME ACTIVITY


relating protein amounts & enzyme activity

1 (international) UNIT of ENZYME ACTIVITY that amount of protein which converts 1 micromole of substrate to product per min at 250C at optimal pH UREASE - 1 unit (IU) will liberate 1.0 mole of ammonia from urea per minute at pH 7.0 at 25C [equivalent to 1.0 I.U.] 1 UNIT of SPECIFIC ACTIVITY the number micromoles converted per min per mg protein i.e., Units (as above) of enzyme activity per mg protein 1 UNIT of MOLECULAR ACTIVITY number of units of enzyme activity per mole

Protein Purification

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