SALTING IN

1. T A H A N I A L S H E H R I
Salting in , salting out and
dialysis

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

3. 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.

4. (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.

5. ** 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 .

6. 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?)

8. 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.

9. 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