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

2. 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 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 (H 2 O) 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 (H 2 O) 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 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 H 2 O molecules interacting with surface groups, in order to stay in aqueous solution (hydration). – Salting out usually uses increasing concentrations of ammonium sulfate [(NH 4 ) 2 SO 4 ] to compete with the protein groups for the available H 2 O. – 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 H 2 O molecules interacting with surface groups, in order to stay in aqueous solution (hydration). – Salting out usually uses increasing concentrations of ammonium sulfate [(NH 4 ) 2 SO 4 ] to compete with the protein groups for the available H 2 O. – 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")

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

6. Why choosing (NH 4 ) 2 SO 4 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 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 & Materials - Saturated solution of (NH 4 ) 2 SO 4 - Finely powdered crystalline (NH 4 ) 2 SO 4 - Biuret reagent - Blood serum sample - Test tube stand with a set of test tubes - Filtering funnel - Filter paper - Saturated solution of (NH 4 ) 2 SO 4 - Finely powdered crystalline (NH 4 ) 2 SO 4 - Biuret reagent - Blood serum sample - Test tube stand with a set of test tubes - Filtering funnel - Filter paper

10. 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. 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 agentSulfte ammonium solution saturation degree Fraction of blood serum proteins to be ppt