The document discusses the process of protein salting-out, where high concentrations of salts like ammonium sulfate are used to precipitate proteins out of solution. At low salt concentrations, solubility increases due to screening of charge-charge interactions, but at high concentrations the salt competes for water molecules needed to solvate proteins, removing their solvation sphere and causing precipitation. Fractional precipitation uses increasing ammonium sulfate concentrations to sequentially precipitate different proteins based on their individual solubility profiles.

1. Protein Salting-outProtein Salting-out
IUG,2012
TMZ
IUG,2012
TMZ

2. Salting in / Salting outSalting in / Salting out
• Salting IN
• At low concentrations,
added salt usually increases
the solubility of charged
macromolecules because
the salt screens out charge-
charge interactions.
• So low [salt] prevents
aggregation and therefore
precipitation.
• Salting IN
• At low concentrations,
added salt usually increases
the solubility of charged
macromolecules because
the salt screens out charge-
charge interactions.
• So low [salt] prevents
aggregation and therefore
precipitation.
• Salting OUT
• At high concentrations
added salt lowers the
solubility of macro-
molecules because it
competes for the solvent
(H2O) needed to solvate the
macromolecules.
• So high [salt] removes the
solvation sphere from the
protein molecules and they
come out of solution.
• Salting OUT
• At high concentrations
added salt lowers the
solubility of macro-
molecules because it
competes for the solvent
(H2O) needed to solvate the
macromolecules.
• So high [salt] removes the
solvation sphere from the
protein molecules and they
come out of solution.

3. General protocol for protein purificationGeneral protocol for protein purification
• Taking the intact Tissue.
• Homogenisation
• Getting rid of debris and insoluble stuff
• Precipitation of protein with the salt( salting –
out)
• Getting rid of salt excess by dialysis
• Further purification by column and ion exchange
chromatography ,
• Finding out the exact molecular weight by
Column chromatography and by SDS-Gel-
electrophoresis
• Taking the intact Tissue.
• Homogenisation
• Getting rid of debris and insoluble stuff
• Precipitation of protein with the salt( salting –
out)
• Getting rid of salt excess by dialysis
• Further purification by column and ion exchange
chromatography ,
• Finding out the exact molecular weight by
Column chromatography and by SDS-Gel-
electrophoresis

4. – Proteins require H2O molecules interacting with
surface groups, in order to stay in aqueous solution
(hydration).
– Salting out usually uses increasing concentrations of
ammonium sulfate [(NH4)2SO4] to compete with the
protein groups for the available H2O.
– Like all purification methods, salt fractionation has to
be worked out empirically for each protein of interest
– Every protein in the solution has its own solubility
limits in ammonium sulfate, independent of the
other proteins in the mixture.
– Proteins require H2O molecules interacting with
surface groups, in order to stay in aqueous solution
(hydration).
– Salting out usually uses increasing concentrations of
ammonium sulfate [(NH4)2SO4] to compete with the
protein groups for the available H2O.
– Like all purification methods, salt fractionation has to
be worked out empirically for each protein of interest
– Every protein in the solution has its own solubility
limits in ammonium sulfate, independent of the
other proteins in the mixture.
Fractional Precipitation ("salting out")Fractional Precipitation ("salting out")

5. In general, solubility of a given protein is independent of othersIn general, solubility of a given protein is independent of others

6. Why choosing (NH4)2SO4 for precipitation?Why choosing (NH4)2SO4 for precipitation?
• Has a wide range of
application
• Very effective to ppt out
water soluble proteins.
• These ions have stabilizing
effect on protein
• You can do sequential ppt of
your desired protein
depending upon its molecular
weight.
• Proteins are readily stored as
ammonium sulfate ppt.
• Has a wide range of
application
• Very effective to ppt out
water soluble proteins.
• These ions have stabilizing
effect on protein
• You can do sequential ppt of
your desired protein
depending upon its molecular
weight.
• Proteins are readily stored as
ammonium sulfate ppt.

7. In-lab experimentIn-lab experiment
• Principle
The experiment is based on the fact that ammonium
sulfate neutralizes the charge on the protein
molecules, and induces their dehydration-resulting in
a protein precipitation (salting-out).
• Principle
The experiment is based on the fact that ammonium
sulfate neutralizes the charge on the protein
molecules, and induces their dehydration-resulting in
a protein precipitation (salting-out).

8. • Half-saturation
causes ppt of gloubulins (less hydrophilic,
larger molecular mass, compred to albumins)
• Complete saturation
causes ppt of albumins
• Half-saturation
causes ppt of gloubulins (less hydrophilic,
larger molecular mass, compred to albumins)
• Complete saturation
causes ppt of albumins

9. Reagents & MaterialsReagents & Materials
- Saturated solution of (NH4)2SO4
- Finely powdered crystalline (NH4)2SO4
- Biuret reagent
- Blood serum sample
- Test tube stand with a set of test tubes
- Filtering funnel
- Filter paper
- Saturated solution of (NH4)2SO4
- Finely powdered crystalline (NH4)2SO4
- Biuret reagent
- Blood serum sample
- Test tube stand with a set of test tubes
- Filtering funnel
- Filter paper

10. ProcedureProcedure
1. 20 drops of blood serum are transferred in test tube.
2. An equal volume of sat., ammonium sulfate solution is
added (half-saturation soln.).
3. Let soln stand for 5 min, and then filter the precipitate off.
4. A powdered ammonium sulfate is added to the filtrate by
small portions until no more visible dissolution of the salt
added occurs (complete sat.,).
5. Note albumin precipitate to form. Filter the precipitate off.
6. Check the filtrate for the absence of protein by applying
the biuret test.
1. 20 drops of blood serum are transferred in test tube.
2. An equal volume of sat., ammonium sulfate solution is
added (half-saturation soln.).
3. Let soln stand for 5 min, and then filter the precipitate off.
4. A powdered ammonium sulfate is added to the filtrate by
small portions until no more visible dissolution of the salt
added occurs (complete sat.,).
5. Note albumin precipitate to form. Filter the precipitate off.
6. Check the filtrate for the absence of protein by applying
the biuret test.

11. Salting-out agent Sulfte ammonium solution
saturation degree
Fraction of blood serum
proteins to be ppt