unable to ppt. protein using ammonium sulphate
rcwieg at ccmail.monsanto.com
Fri Nov 18 10:07:32 EST 1994
In article <BBE315406CC at microb.umd.edu>, SABLAB at MICROB.UMD.EDU ("S.A.
Benson Lab") wrote:
> 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. I'm sure that the
> concentration of the protein is at least about 15ug/ml and I can see
> a band on SDS gel when I loaded 10ul of the sample. I heard that
> usually 45% of it will do the job! and I don't know what the story is
> about my situation. Would someone who has an idea of this
> situation gives me some advices please? and thanks in advance!
Not all proteins precipitate with AmSO4. The concentration to be used must
be empirically determined for each protein, and will typically range from
20-80% saturation. The efficiency will tend to be lower at lower protein
concentrations (yours is pretty low). If your protein stays up at high
AmSO4 you actually have a very nice purification step, if not a useful
method for concentration.
Try taking the AmSO4 concentration higher--to saturation if necessary to
see if your protein will come out. If it doesn't you simply need a
different concentration method. (You are doing this on ice, aren't you?)
mailto::rcwieg at ccmail.monsanto.com
More information about the Methods