tysondr at sluvca.slu.edu
Mon Nov 18 13:49:30 EST 1996
Haralt Leiter wrote:
> Hi everybody !
> I´m a poor PhD Student (my supervisor hasn´t paid me) purifying a
> fucosyltransferase with an estimated (SDS-PAGE) molecular weight of at
> least 50kDa. During ultrafiltration the entire enzyme-activity passes
> through the membrane (cutoff 30000). Has anybody observed a similar
> effect or can offer a reasonable explanation ?
> I use a 25mM Tris/HCl pH7,3
> 0,1% Triton X-100
> 10% Glycerol
> 0,02% NaN3 buffer and the applied protein solution contains several
> hundreds of micograms.
> Thanks for any answers in advance
> BTW, I have already tried membranes of different companies
It's POSSIBLE that your protein is actually smaller than 30,000 Da
and simply runs slower on SDS-PAGE. Have you tried a lower MW cutoff
say 5,000 Da to be sure its still there? Also, are you sure about
the correlation of the band seen on SDS-PAGE and your activity (i.e.
is it possible that the enzyme is actually a smaller protein that
simply copurifies with the >50 kDa protein)?
Ph.D. Candidate in Cell and Molecular Biology
Saint Louis University
tysondr at sluvca.slu.edu or darren at primary.net
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