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Purification techniques were done by exploiting the properties by which
proteins differ from one another (Including stability, solubility, charge,
hydrophobicity, size and affinity).
Stability – By altering the temperature (different protein denatures at
Solubility – Ammonium Sulphate precipitation.
Charge – Use of Ion-exchange resins.
Size – Size exclusion chromatography or Gel permeation chromatography
Affinity – Affinity chromatography
Hydrophobicity - Hydrophobic interaction chromatography
- Column used – Phenyl sepharose
- Hydrophobic residues of amino acids binds to the phenyl groups.
Proteins differ – Charge, polarity and hydrophobic amino acids that they
display on their surfaces.
Charged and polar groups on the surface are solvated by water molecules,
making the protein molecules soluble – Hydrophobic residues are masked by
water molecules that are adjacent to these regions.
Proteins precipitate differentially from solution on the addition of species such
as neutral salts or organic solvents.
Salt fractionation is frequently carried out by ammonium sulfate precipitation.
As increasing salt is added to the protein solution, the salt ions are solvated by
water molecules in the solution.
As the salt concentration increases, the freely available water molecules that
can solvate the ions become scarce.
At this stage, those water molecules that have been forced to be in contact with
the hydrophobic groups on the surface of the protein are the next most freely
available water molecules and these are therefore removed to solvate the salt
molecules, thus leaving the hydrophobic patches exposed.
These hydrophobic patches cause proteins to aggregate by hydrophobic
interactions resulting in precipitation.
The first proteins to aggregate are those with most hydrophobic residues on the
surface followed by those with less hydrophobic residues.
The aggregates formed are mixture of more than one protein.
As many proteins are precipitated over a narrow range of salt concentrations,
this makes the technique simple for enriching protein of the interest.
Practical example for fractional precipitation using Ammonium sulphate
As increasing amounts of ammonium sulfate are dissolved in protein
solution, certain proteins aggregate and start precipitate out of the solution.
Increase in the salt strength further precipitates different proteins out.
A controlled pilot experiment will be performed, where the percentage of
ammonium sulfate increases step-wise from 10%, 20%, 30% and so on.
The resultant precipitate is removed by centrifugation and redissolved in
buffer and analyzed for protein of interest.
The original homogenate was made 45% in ammonium sulfate and the
precipitate recovered and discarded.
The supernatant was made 70% in ammonium sulfate, the precipitate is
collected and re dissolved in buffer, supernatant being discarded.
Role of Ammonium Sulphate
It is the most used salt due its high solubility in water.
It stabilizes most proteins in solution and helps reduce the lipid content of
Removes up to 50% of contaminating protein, and therefore reduces the load
for subsequent chromatography.
Most precipitated proteins retain their activity and native conformation, and
can be redissolved easily.
Principle in nut-shell
At low concentrations, the presence of salt stabilizes the various charged
groups on a protein molecule, thus attracting protein into the solution and
enhancing the solubility of protein. This is commonly known as salting-
As the salt concentration is increased, a point of maximum protein
solubility is usually reached. Finally, protein starts to precipitate when
there are not enough water molecules to interact with protein molecules.
This phenomenon of protein precipitation in the presence of excess salt is
known as salting-out.