[Protein-analysis] Re: purification & sequencing
Dr Engelbert Buxbaum
engelbert_buxbaum at hotmail.com
Mon Mar 13 04:01:05 EST 2006
chetanchandra josh wrote:
> I hv isolated a caseinolytic protease but may have residual contamination of casein.
> I used the media (M9 without amino acids and casein as only carbon source).
> I performed salting process And dialysis.
Salt (presumably ammonium sulfate?) precipitation is a very crude
purification method and should be complemented by something more
selective. SEC and IEC are examples.
If you want to distinguish your protease from casein on SDS-gels, use
one lane with a casein standard.
Another option is to use zymograms, that is, you use the enzymatic
activity of your protease to get a specific staining reaction in the
gel. Chromogenic substrates for proteases are commercially available.
After SDS-PAGE enzymes are usually inactive (although there are
exceptions), but after native PAGE activity is often maintained. You can
try if your protease is activ in the presence of CTAB, a mild,
positively charged detergent. If so, you can do zymograms in CTAB-gels,
the advantage over native gels would be that separation is by molecular
weight, as with SDS.
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