(no subject)

Darren Tyson 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
> Haralt
> 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)?

Darren Tyson
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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