Lab 6

Salt fractionation

In aqueous environments salt ions interact strongly with their

complementary charge on a water molecule

If the concentration of salts is high enough the ions will effectively

compete for the water molecules bound to the proteins in solution.

Without water molecules to associate with, the proteins will seek

complementary charges on other proteins, thus aggregating together

and precipitating out of solution

A. Ammonium sulphate precipitation

Advantage: concentrates your proteins

Ammonium salts precipitates out different proteinsat different concentrations, depending on physical properties of the protein

IgG is quite hydrophobic so it precipitates out before manyother proteins.

B. Caprylic acid (Octanic acid)

Weak acidic buffer plus the addition of short chain fatty acids,(caprylic acid) will precipitate most serum proteins (not IgG)

Inexpensive, fast

CH3(CH2)6COOH

C. Ion exchange chromatographyThe DEAE part of the anion exchange matrix DEAE-Sephacel

DEAE has a net positive charge, so it binds proteins that have a netnegative charge (anions)

The net charge on a protein will change in response to the pH in its

environment. Each protein has a distinct isoelectric point and thus has

a distinct pH at which it will no longer bind an ion exchange column

Usually in ion exchange

chromatography the proteins

are bound to the

matrix and then pH is

gradually altered so that

proteins with different

isoelectric points are eluted

off the matrix at different

points in the pH

gradient. The same can be

achieved by increasing the

salt concentration as shown

here

DEAE cellulose: separates on basis of charge

D. Protein A or Protein G columns

Especially good for mAb purification from ascites i.e.mouse IgG1

Very species and isotype specific

Principles of dialysis

Tubing has a molecular weight cut-off that indicates the size ofthe pores or holes in the membrane.

All proteins, and contaminants that are smaller than the holes will leak out.

Water often will leak in to dilute the salts.