Salt fractionation environments salt ions interact strongly with thei ary charge on a water molecule centration of salts is high enough the ions will ef r the water molecules bound to the proteins in solu ter molecules to associate with, the proteins will ary charges on other proteins, thus aggregating tog itating out of solution
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Lab 6 Salt fractionation In aqueous environments salt ions interact strongly with their complementary charge on a water molecule If the concentration of.
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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.