Separation by Superose 6 gel filtration

WS via (by novalidaddress from
Thu Nov 11 16:05:38 EST 2010


my first thought was that you possibly have some sort of protease in
your prep that becomes active when the inhibitors are removed by the
desalting process (as small molecules migrate slower that large
molecules and you probably won't include inhibitors in the column
buffers due to their large volume).

But I understand it correctly that the bands occur in *all* fractions
you are collecting from the column (even in an empty run)? So,
possibly that what you see on the gel / western is not protein, but
some sort of leakage that comes from the column. If you don't have the
opportunity to identify the nature of the band by mass spectrometry,
you could do some simple experiments to get an idea what you might
have; either your gel is degraded and is leaking some sort of possibly
polysaccharides or there is a contamination on the column (when it has
been used for something else previously or not been stored properly).
You didn't write about the nature of your sample; is there a
possibility that it contains enzymes that may degrade the sepharose?

a) try to thoroughly clean the column as described in the manual. If
the problem persists,
b) try to digest the eluate with proteases, eg trypsin. if there is
protein, it should become degraded
c) add some (sodium) metaperiodate in order to oxidize diols. If your
band gets degraded, it is a polysachharide.

You also might simply ask the technical service of your supplier for
help. They should know their stuff. Other possibilities are to snatch
a different batch from the neighbor lab and check if it behaves the



PS is gel filtration the first step of your purification process?

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