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Lab Session 3. Protein Salting-out . IUG,2012 TMZ. 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

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lab session 3

Lab Session 3

Protein Salting-out

IUG,2012

TMZ

salting 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 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 in / Salting out
general protocol for protein purification
General 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

slide4

Fractional Precipitation ("salting out")

  • 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.
why choosing nh 4 2 so 4 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.

in lab experiment
In-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).

slide8

Half-saturation

causes ppt of gloubulins (less hydrophilic, larger molecular mass, compred to albumins)

  • Complete saturation

causes ppt of albumins

reagents materials
Reagents & 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

procedure
Procedure

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.