unable to ppt. protein using ammonium sulphate

Jim Owens jow at helix.nih.gov
Fri Nov 18 08:57:26 EST 1994


In article <BBE315406CC at microb.umd.edu> S.A. Benson Lab,
SABLAB at MICROB.UMD.EDU writes:
>I have a problem trying to precipitate a purified protein, I have 
>used almost 60% of ammonium sulphate and actually the solution is 
>saturated and I still don't see any cloudiness. 

This may not help your problem, but it may clear up a misconception.  

When one uses ammonium sulphate for protein precipitation, the per cent
is that of saturation, generally at zero to four degrees C.  In other
words, 100% ammonium sulphate is a saturated solution.  Usually one makes
up a solution at a high temperature until no more ammonium sulphate
dissolves and then cools in an ice bath or cold room.  The supernate is
saturated (100%) ammonium sulfate.

As for your problem, it may be that since this precipitation is supposed
to occur at 45% of saturated that you have salted in the protein of
interest.  Try dialysing to get rid of excess salt.  According to the
Handbook of Chemistry & Physics 70.6 g of ammonium sulphate will dissolve
in 100 cc of water.  So you should dialyse against about 1.05 volumes of
water.  I don't know if your dialysis tubing can take the osmotic
pressure.  I just suggested dialysis to keep the volume down.  If the bag
breaks, you will just have a larger volume to centrifuge.

Good luck,

Jim Owens



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