unable to ppt. protein using ammonium sulphate

[Roger Wiegand]

In article <BBE315406CC at microb.umd.edu>, SABLAB at MICROB.UMD.EDU ("S.A.
Benson Lab") wrote:

> Hello!
> 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?)

-- 
Thanks,
Roger

mailto::rcwieg at ccmail.monsanto.com