I can't concentrate!

[rpgrant at molbiol.ox.ac.uk]

In article <Pine.3.89.9403251526.A22537-0100000 at fox.cce.usp.br>,
szeinfel at FOX.CCE.USP.BR (Rafael N Szeinfeld) writes:

|>On Thu, 24 Mar 1994, Brian Ellis - lab wrote:
|>
|>> Here is problem that i haven't encountered before.  After FPLC i
|>attempt to 
|>> concentrate the fractions in centricon/centriprep tube (MW cutoff
|>10K), it 
|>> appears that my protein precipitates out of solution.  Has anyone
|>else 
|>> encountered this problem.  The protein is 78 kD in size and is found
|>in native 
|>> state as a tetramer.  
 
|>	I'm using centricon to concentrate a trypsin derivated from it 
|>reaction with a free radical and after concentration we can't find our
|>protein. At first we thought that the membrane was bad but now we
|>think 
|>that our protein in suffering autolysis, could it be your case ?
|>

	I had rather a bizarre problem with a recombinant protein I was making.
It behaved quite happily until I tried putting it into low salt ready
for Xtallization trials.  After some head scratching, I finally
discovered that I got good concentration and recovery in 100 - 200 mM
salt, but down at 10 - 20 mM salt (and lower) it took an age to
concentrate, and the recoveries were lousy.  So now I concentrate in 200
mM Tris, 20 mM NaCl down to >40 mg/ml protein, take it well away from
any damn centricons, and dilute it to working conc with H2O.  It seems
quite happy like that :-)

	P.S.  A minor contaminant of the prep was found to behave normally and
its relative abundance therefore increased quite dramatically.

--  
Richard P. Grant    <><                rpgrant at molbiol.ox.ac.uk
Sir William Dunn School of Pathology   Fax. +44 865 275501
University of Oxford, UK.              Tel. +44 865 275582