Protein ultrafiltration query

D.Lippert lippy at UVic.CA
Fri Nov 15 11:53:17 EST 1996


Haralt Leiter <hleiter at edv2.boku.ac.at> wrote:

>Sorry, I earlier forgot a subject line:

>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

If you are certain that you are handling the membrane properly (not
scratching it or something) then I would suggest using a smaller
molecular weight cut-off.  This will most likely solve your problem.
The estimate you get from SDS-PAGE is just that - an estimate.  Many
proteins do not migrate at their true molecular weight.  This is
especially true for glycoproteins that tend to appear much larger than
they truly are.  It may also be that your molecule has an unusually
large proportion of hydrophilic amino acids.  This can lead to low
binding of SDS, which results in an overestimate of MW by SDS-PAGE.
Incidentally, I work on a 55kDa enzyme that appears to be ~100kDa on
SDS-PAGE.  Hope this helps.

Lippy




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