T A H A N I A L S H E H R I

Salting in , salting out and dialysis

Solubility of proteins

Solubility:

• very big differences: structural (fibrilar) and membrane proteins are practically insoluble, some globular proteins are extremely soluble

Salting in – Salting out

Salting-in effect: ions shield charges and decrease protein-protein electrostatic interaction;solubility increase!

Salting-out effect: ions take‘all’ water; solubility decrease!

Used to selectively precipitate proteins, often with (NH4)2SO4 which is cheap, effective, does not disturb structure and is very soluble.

(NH4)2SO4

Used to selectively precipitate proteins, because it is:

cheap

effective

does not disturb structure (no harmful effect)

Relative freedom from temperature effects

very soluble.

** Solubilities of Proteins

Salting In

Addition of salt at low ionic strength can increase solubility of a protein by

neutralizing charges on the surface of the protein, reducing the ordered water

around the protein and increasing entropy of the system.

Salting out (Can be used for Fractionation)

If the concentration of neutral salts is at a high level (>0.1M), in many

instances the protein precipitates.

This phenomenon apparently results because the excess ions (not bound

to the protein) compete with proteins for the solvent. The decrease in solvation

and neturalization of the repulsive forces allows the proteins to aggregate and

precipitate. This effect is called "salting out".

**other def, for me .

The effect of salt on different proteins may differ:

Certain proteins precipitate from solution under conditions

In which others remain quite soluble.

Once the protein is precipitated (not denatured) –

can separate by centrifugation pellet

can be redissolved in buffer for further purification

ⓆWhich protein will ppt first? (hydrophobic or hydrophilic?)

Dialysis

Passage of solutes through a semi-permeable membrane.

Pores in the dialysis membrane are of a certain size.

Protein stays in; water, salts, protein fragments, and other molecules smaller than the pore size pass through.

Dialysis

• Following a salting-out step, the solution will contain a

high concentration of salt that can be disruptive to

subsequent chromatographic steps.

• The salt can be removed by dialysis – dialysis tubing

has pores with a specific molecular weight cut-off that

allows smaller molecules (salt) to pass.

Exchange buffer

> 3 times

Buffer– large volume

Dialysis tubing with protein and high salt