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.
More information about the Proteins