Thanks for the good suggestion Ina. Freezing in glycerol is probably
one of the best ideas raised so far. The beads can be quickly washed
after bringing to 4decC, to remove glycerol.
We don't freeze the protein and bind it to the beads later, because we
already have so much protein and bead available each time we do the
expression (we don't like to waste it).
- For free protein, we have to elute from the GSH-agarose with GSH, then
dialyse to remove GSH, then freeze, then re-apply to the column - works
ok, but we lose a number of the fusion proteins on dialysis (not all of
them, some behave well), and it takes a couple days.
- We don't freeze the bacterial pellet very often, as this requires
homogenisation, centrifugation, binding the column, washing, then
mini-gels to verify the amount of protein bound to the column - again,
works ok, but takes a couple of days to be able to end up with exactly
the same amount of protein bound/ul bead. We like to do all this work
*once* (or once-in-a-while), then use the beads for pull-downs. Its
*far* easier to have a constant supply of beads that we have
characterised already, than to redo it for each experiment.
Phil R
CMRI, Sydney
nospam at our.site wrote:
> 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
