5. Ammonium Sulphate Precipitation


Isolation of Immunoglobulin rich fraction




Different proteins can be gradually precipitated out from their aqueous solutions    using highly soluble salts such as ammonium sulphate. As the concentration of the salts is increased they compete with proteins for water molecules and lead to their gradual precipitation based upon their requirement for water molecules to be in the soluble form. By convention the final concentration of ammonium sulphate required for precipitation is expressed in terms of percentage of saturation. Immunoglobulin rich fraction can be precipitated out from serum by 33% saturation with ammonium sulphate.


B.Material :


a. Serum.


b.0.01 M SPB, pH 7.2       (Appendix - I)


c.Saturated ammonium sulphate solution.


  Add about 75 g of ammonium sulphate to 100 ml distilled water and stir. Heat the solution and filter through Whatman No.1 filter paper while it is hot. After the solution comes to room temperature, adjust the pH to 6.8 using concentrated ammonium hydroxide and store. Ammonium sulphate crystals should be formed in the container after few hours.


d. Spectra / Por dialysis tube.


e.Magnetic stirrer.


f.Refrigerated centrifuge.


C. Method :


1.Add  5 ml  saturated  ammonium sulphate  solution gradually to 10 ml of serum under constant stirring at ice cold temperature.


2.Keep the suspension in ice for 15 minutes with occasional stirring.


3.Centrifuge at 3000 RPM for 30 min at 4 0C.


4.Discard the supernatant and wash the precipitate twice with 40% ammonium sulphate solution  ( Mix  thoroughly with  glass  rod and centrifuge at  3000 RPM for 30 min at  4 0C).


5.Reconstitute the precipitate in small volume of 0.01 M SPB (pH 7.2) and dialyse against the same buffer for 24 hr with frequent changes of buffer.