Freezing GSH-agarose for "pull-down fever (PDF)"

Fred dynamin at hotmail.com
Wed Mar 1 20:10:04 EST 2000


We all know that freezing agarose or sepharose beads in aqueous
solutions fractures them and ruins them for gel filtration.  I want know
if they actually break apart into small pieces.  In other words, can
they still be used for pull-downs.

We attach GST-pusion proteins to GSH-agarose, and need to keep the beads
for a long time for a frenzy of pull-downs we are doing.  We find they
get bacterial contamination or lose activity after a month or so at
4degC.  The purified protein off the beads are all stable when frozen
for a year.  So we are thinking of freezing the beads with the fusion
protein attached.  The beads are only there for the pull-down part of
the assay, so cracks and fractures are not a problem in theory.  (We
tried azide, to disastorous effect on our activity).  Anybody got any
comments on whether this might work?  It will save us expressing the
same proteins over and over...

A related question.  How does one dry these beads in a way that prevents
the cracking?  I presume we would add sucrose or trehalose? or some
such.  What? How much?  Is a lyophilyser ok?  This might also solve our
problem, if our protein reconstitutes functionally.

Phil R	Sydney




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