wolfsc at ibms.sinica.edu.tw
Fri Dec 7 10:49:51 EST 2001
do you think that using a bifunctional (i.e. crosslinking) aldehyde like
glutardialdehyde could improve fixation?
> Sergio wrote:
> > Hi all,
> > we have purified a hybrid protein (MBP-R), getting a nice yield. However, when
> > we cut this one to split the hybrid protein into MBP and R, we found two
> > problems:
> > 1 - low cutting efficiency of the FactorXa, whereas it works perfectly on
> > another MBP-proteins we've purified in our lab. We think the R protein is
> > getting folded and covering the FactorXa cutting site. We have added SDS at very
> > low concentration (0,005%) trying to relax the folding, but we haven't been
> > lucky so far. Suggestions??
> Try using more Xa, or longer digestion times. For one protein of mine, I
> had to use a 1:25 ratio of protease to fusion protein, and digest over
> > 2 - Although we can see the non-cutted MBP-R and some free MBP in a Coomassie
> > stained PAGE, we don't see the R protein band. This one is expected to be a 9300
> > Da protein. Since we've been told that some proteins aren't well stained with
> > Coomassie, we are thinking on doing a silver staining on the Coomassie stained
> > gels. Does anybody have a good protocol for this?.
> in many cases, small proteins are not fixed well in the gel, and readily
> diffuse out during the staining process. We had the same problem, until
> we switched to a different formulation of the coomassie staining
> solution. Using one with formaldehyde provides a much better fixation,
> and a much higher sensitivity for small proteins. The formulation is
> described in Steck et al, 1980, Anal. Biochem. 107:21-24.
> Staining solution:
> 18ml EtOH, 42ml Water, 10ml formaldehyde (from 37% stock), 0.08g Coomassie
> (as always with coomassie solutions, do NOT reuse!)
> destaining solution:
> 250ml EtOH, 750ml water, 10ml formaldehyde
> staining takes approximately the double time, we used 1h for minigels.
> Replace with destaining solution as usual, until bands are well visible.
> In contrast to normal coomassie staining procedures, the sensitivity
> increases quite significantly when you store the gel in destaining
> solution plus 1/10 volume staining solution, overnight in the fridge.
Dr. Wolfgang Schechinger
Institute of Biomedical Sciences
Academia Sinica, Taipei, Taiwan R.O.C.
e mail wolfsc at ibms dot sinica dot edu dot tw
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