Salting out is a method of separating proteins based on the
principle that proteins are less soluble at high salt
concentrations. The salt concentration needed for the protein to
precipitate out of the solution differs from protein to protein.
Addition of salt at low ionic strength can increase solubility of a
protein by neutralizing charges on the surface of the protein,and
this is called salting in.
When the salt concentration is increased some of the water
molecules are attracted by the salt ions, which decreases the
number of water molecules available to interact with the
charged part of the protein. As a result of the increased demand
for solvent( water ) molecules, the protein-protein interactions
are stronger than the solvent-solute interactions; the protein
molecules will tend to coagulate ,aggregate, and precipitate and
this is the salting –out phenomenom.
Gel Chromatography is a type of partition chromatography .
Partition Chromatography ; In partition chromatography the solute
molecules distribute themselves between two immiscible phases . It
basically involves the separation of a mixture of molecules due to
differences in their distribution coefficients between two different
phases.
In gel chromatography one phase is the mobile phase the second is the
stationary phase. The stationary phase is composed of inert beads
that contain pores that span a narrow size range.
The mobile phase is the buffer that is added continuously to the column
to elute the samples.
The solute molecules are separated according to their molecular size
mainly , solute molecules will travel in the column indifferent rates
very large molecules will be excluded totally from the beads (cannot
pass through the bead pores ) thus they will be the first to be eluted
. Very small molecules will be totally included in the beads thus they
will be retarded by the beads and thus will be the last to be eluted.

Thus the solute molecules will be eluted in the following order
large molecules first followed by smaller molecules then the
smallest will eluted last.
 Example; Consider the separation of the following mixture
-Glutamate dehydrogenase ( molecular weight 290000) .
-Lactate dehydrogenase (molecular weight 140000).
-Serum albumin (molecular weight 67000).
-Ovalbumin (molecular weight 43000).
-Cytochrome C (molecular weight 12400).
With Bio-gel-P-150.fractionation range (15000-150000).

When the protein mixture is loaded on the column , the first protein
to be eluted is Glutamate dehydrogenase which is totally
excluded from the beads since its MW is above the upper
fractionation limit.

Cytochrome C is the last to be eluted from the column since its
MW is below the lower fractionation limit so it will enter the
pores of the beads and thus be retarded in the column and be
eluted last.The rest of the protein molecules will be eluted in the
order of decreasing MW.

So the order of elution is ;
1- Glutamate dehdrogenase.
2-Lactae dehydrogenase.
3-Serum albumin.
4-Ovalbumin.
5-Cytochrome C.
Size exclusion chromatography
Gel filtration chromatography
 Contains porous beads
 Separates according to size and shape
 Larger proteins excluded
from the small pores
 Quaternary structure determination,
& Mr estimation using
a standard curve
(log Mr vs elution volume)
Ⓠ Fibrous proteins
Spherical vs rod-shaped proteins
Gel Electrophoresis

Since proteins are charged macromolecules they can be
separated based on their charges.
In Gel electrophoresis charged molecules are separated on the basis
on differences in their charge and size . The charged molecules
will migrate across the gel towards either the cathode or the
anode according to the net charge on the molecule , negatively
charged molecules will migrate towards the anode while
positively charged molecules will migrate towards the cathode .
The direction of the molecules (towards anode or cathode)
depends on the type of charge on the molecule .
The distance migrated by the molecules depends on the magnitude
of the charge and the MW of the molecule .The higher the charge
magnitude the larger the distance migrated.
The larger the MW of the molecule the shorter the distance
migrated.
passage of molecules according to their size and shape.

The net charge on the protein molecule depends on the proteins
pI and the pH of the separation media .

Example ;
A mixture of the following proteins ;
- Protein( A) pI= 6.0 , MW = 14000.
-Protein (B ) pI = 4.0 , MW = 35000.
-Protein (C) pI = 6.0 , MW = 55000.
Protein (D) pI = 5.0 , MW = 23000.
The pH of the separating media (buffer) is pH= 5.0.
Thus the net charge on the molecules will be ;
Protein (A) pH < pI thus net charge on the protein is +ve .
Protein (B) pH > pI thus net charge on the protein is –ve.
Protein (C) pH < pI thus net charge on the protein is +ve.
Protein (D) pH = pI thus net charge is zero.

Thus protein (A) and (C) are +vely charged so they will migrate
towards the cathode , but protein A will migrate to a larger
distance since it has a lower MW.

Protein (B) is –vely charged thus it will migrate towards the
anode .

Protein (D) has a zero net charge thus it will not move from the
origin.
Proteins move in the
electric field. Their
relative speed depends on
the charge, size, and
shape of the protein
Immediately after electrophoresis proteins in the gels are
precipitated by either adding alcohol containing solutions
or strong acids (e.g. TCA).
Protein are often stained by Coomassie Blue dye or by
photography-like treatment with AgNO3 (silver staining)
There are many other stains available (e.g. Stains-all,
fluorescence probes etc.)
Silver staining is usually
10-100 times more
sensitive than Coomassie
Blue staining, but it is
more complicated.
Faint but still visible
bands on this gel contain
less than 0.5 ng of
protein!