Protein Purification Techniques

All work done at 4oC to minimize degradation.
1- preparation of the protein solution
With appropriate buffer, must first disrupt cells by mechanical
homogenization with either detergent and/or enzyme treatment.
Use a centrifuge to separate into pellet and supernatant.
2- fractionation (relies on protein solubility differences)
Use ammonium sulfate (salt) --> interferes with noncovalent bonds between protein
and other molecules. Remember solubility is based upon interactions of molecule
with water molecules via hydrogen bonds.
Different proteins precipitate out at different [salt].
Use centrifuge to remove precipitated protein --> resuspend in buffer.
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Use dialysis to change out solvent and get rid of NH4SO4.
3- chromatography
Further fractionates proteins based upon protein’s interaction with matrix.
Most commonly used is column chromatography.
Uses beads or cellulose fibers.
Protein solution is washed through column.
Eluate collected and assayed for protein.
There are three types of column chromatography:
1) ion-exchange (anion or cation)
Separates based upon protein charge.
Elute by changing [salt].
2) gel filtration
Uses porous resin.
Separates based upon protein size.
3) affinity chromatography
Attach ligand to matrix. Can be substrate, antibody, etc.
Eluate using high [ligand] or high [salt].
Results in 1000-10,000 fold purification.
4- electrophoresis
Separates proteins based upon migration in an electric field.
PAGE - polyacrylamide gel electrophoresis
Uses acrylamide as gel matrix.
Separates based upon size and charge (buffer is slightly basic, so most
proteins have negative charge).
SDS-PAGE - sodium dodecyl sulfate polyacrylamide gel electrophoresis
Uses SDS and 2-mercaptoethanol.
Separation based upon size only.
For both, must stain gel to visualize proteins.
Bands can be cut out of gel, protein electroeluted, and purified.
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