Protein purification problem

P.C. PC at nospam.edu
Sat Mar 13 13:45:20 EST 2004


well, it does sound like it could be not a protein at all, I agree with
H.S.R. But assuming it is a protein, one thing that comes to mind is
boiling.

Some proteins do not tolerate boiling. One example I had - misterious
dissapearances of a recombinant viral capsid protein. I had tons and
tons of it, but somitimes I would see nothing just some weak ugly smears
upwards and some stuff in the stacking gel. The solution was to avoid
boiling and instead to leave it at room temp for 10-20 min in the SDS
sample buffer (it is enough to reduce cystein residues in most cases).

Peter

gilbertjack at hotmail.com wrote:
> I am currently working on the most difficult project I have ever seen and have finally been forced to take the plunge and ask for outside assistance.
> 
> 
> 
> This protein is a novel type from bacteria. Size exclusion chromatography suggests it to be 35-45kDa. It does not stain by coomassie, silver stain or any traditional techniques on SDS-PAGE. However, it is stained by Stains-All, but this shows it to be stuck at the stacking/resolving gel interface. 
> 
> 
> 
> We have tested for amino-sugars and found none. 
> 
> 
> 
> Does anyone have any idea why a protein would not travel through a polyacrylamide gel at all?
> 
> 
> 
> If anyone has noticed anything like this before I would love to speak with you in more detail. 
> 
> 
> 
> Yours sincerely,
> 
> 
> 
> Jack Gilbert
> 
> http://biowww.net/mynews/tree.php?group_name=bionet_molbio_proteins&begin=0





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