help!!! biorad assay vs. BCA assay

Artem Evdokimov AEVDOKIMOZ at cinci.rr.com
Tue Jan 7 18:26:54 EST 2003


<selphydeg at yahoo.com> wrote in message
news:20030107181921.30285.qmail at ww02.hostica.com...
> I am working on a thermophilic protein overexpressed in E.coli.  The first
step in purification is heating.  I used Biorad and BCA assay to determine
the protein concentration, but the result from the two assay is very
different.

All of these assays are approximate. Moreover, the ratios between them won't
be the same for different proteins or even for the same protein measured
under slightly different conditions. Another measurement you might want to
consider is to use theoretical A280 in 6 M guanidine hydrochloride, as
described on www.expasy.ch under ProtParam link. It's neither better nor
worse than all of the other approximate methods.

> I also have trouble purify the protein, and concentrate the protein to
more than 1.5mg/ml.  Does this all point to misfolded protein?

Not necessarily. Some proteins require very specific conditions to be
soluble, detergents, salts, specific pH and whatnot. Follow your nose.

A.G.E.





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