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

Peeter Toomik peeter.toomik at ut.ee
Thu Mar 2 07:20:35 EST 2000



Fred wrote:

> 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.

After freezing you will have a weak gel, but not a broken one. You can try
to freeze and thaw a piece of agarose (electrophoresis) gel and see what
happens.
Or you can try to freeze the gel from glycerol. I hope it will help.

>
> 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

No problems with lyophilising, but you must add ca 30% of sucrose, dextran
or something like.

Peeter Toomik





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