A.Principle:
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.