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
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.
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
Taking the intact Tissue.
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
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.
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).
causes ppt of gloubulins (less hydrophilic, larger molecular mass, compred to albumins)
causes ppt of albumins
- 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
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.