Protein Query

Dr Engelbert Buxbaum engelbert_buxbaum at hotmail.com
Fri Mar 19 09:12:43 EST 2004


gilbertjack at hotmail.com wrote:

> This is an update.
> 
> I am currently working on a bacterial protein which does not seem to
> migrate through an SDS-PAGE or Native-PAGE at all. In SDS-PAGE it get
> stuck at the stacking/resolving gel interface. in Native-PAGE it hardly
> leaves in the Stakcing gel.
> 
> It will only stain with Stains-all, Coomassie and Silver staining
> techniques (and most others i have tried) have no effect.

Such behaviour could mean that you have a protein of very high molecular
weight. If this is the case you need a wide-pore gel for separation, say
2% polyacrylamide. Since such gels would be very soft, add 1% agarose.

It could also be that SDS does not solubilise the protein, you could try
CTAB-PAGE instead (Anal. Biochem. 314 (2003) 70-76). CTAB is a cationic
detergent that can solubilise proteins which are insoluble in SDS, and
can be used to do electrophoresis on proteins like histones, which give
wrong MWs in SDS-PAGE because of their high positive charge.

When preparing your sample, do not boil, but rather heat at 60 degrees
for 30 min, this can help with high MW proteins.



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