Protein Purification Strategies

protease

enzyme that breaks peptide bonds

first step for purification

break the cell to free the protein

#1 Salting Out

theory = water moves from protein to salt causing the protein to clump and form a precipitate.

most soluble has the highest salt concentration and will precipitate first, least soluble, precipitate last

add salt and centrifuge to precipitate out each protein based on solubility. Keep removing precipitate and adding salt to get the least soluble proteins. 

dialysis

to remove the salt that was added during the salting out 

take precipitate and put it in a membrane with freshwater so that the excess salt leaves the proteins

affinity chromatography

column chromatography

• stationary phase = solid silica

• mobile phase = solvent

• 6 histidine molecules at the bottom

1. Flow mixture through column, protein of interest binds to stationary phase (affinity)

2. Keep washing with water to remove the excess solvent

3. Finish washing

4. Elute with Imidazole to remove protein of interest from stationary phase

size exclusion chromatography

separates proteins by size

1. stationary phase is a gel with pores

2. smaller proteins get stuck in pores so take longer to leave

3. larger proteins travel through fast

4. smallest proteins leave last

ion exchange chromatography

proteins attracted to the charge of the stationary phase come out last 

so divides proteins based on charge 

anion exchanger

stationary phase is positive, so anions binds

weak exchanger = DEAE

strong exchanger = Q

cationic exchanger

stationary phase is negative, so cation binds

weak exchanger = CM

strong exchanger = S

SDS PAGE

separates proteins by molecular weight

SDS = sodium dodecyl sulfate (detergent)

binds to proteins and facilitate unfolding to linear molecules and gives it a uniform negative charge

to prepare for gel electrophoresis so that size is only what matters

gel electrophoresis

proteins treated with SDS will move based on size only, they all have same negative charge 

smaller proteins move faster through the gel

depending on how far it goes, you can compare to a standard known values to determine the weight of a protein

electrophoretic mobility

u = Ze/f

voltage = charge of protein/mass

lighter molecules move faster

isoelectric focusing

1. gel is a pH gradient with an electric field.

when the pH = pI, the protein gets stuck at that spot indicating the pI of the protein. 

isolectric focusing with SDS Page in 2D electrophoresis

After separating by PI in the isoelectric focusing separate the molecules by weight on the gel electrophoresis 

So proteins are separated by pI and weight. 

pI is dependent on

AA composition (R groups and end terminals)

in SDS-PAGE the higher the weight

the less it migrated

specific activity =

amt of enzyme performing activity: amt of enzyme

amt of enzyme performing activity =

amt product: time

yield % =

specific activity: total activity of mixture 

how much is the enzyme doing compared to everything goin on in the mixture.