This message has been posted by: 612 right <i.hinners at REMOVE-THIS-TO-SENDicrf.icnet.uk>
Why dont you freeze the protein and bind it to the beads when needed? Or
did I miss something out?
How about adding glycerol i fyou want to store the beads with bound
proteins at -20C?
Kind regards, Ina
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.
>> 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
--
Ina Hinners
ICRF
Secretory Pathways Laboratory
44 Lincolns Inn Fields
WC2A 3PX London
UK
email: I.Hinners at icrf.icnet.uk
