Freezing GSH-agarose for "pull-down fever (PDF)"
peeter.toomik at ut.ee
Thu Mar 2 07:20:35 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.
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
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.
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