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

Antonin Tutter tutter at salk.edu
Sun Mar 5 02:05:26 EST 2000


>
>
> Why -70?  We often find that 20% glycerol frezzes at that temp, vs -20?
> And why rapid freeze?  Does it offer some advantage?

i'm not a chemist or biophysicist, so i can't cite technical arguments for
freezing, but i was always taught to store proteins at -70, and the colder the
better.  as for glycerol, it somehow protects the proteins from the harsh effects
of water crystals - something to do with enhancing hydrophobic interactions.
rapid freezing also aids in protection from crystallization (disorderd
crystals?), but perhaps someone else in netland can supplement this claim with
better scientific explanation.

>
> > i regularly do pulldowns with
> > GST-fusions but i always bind my proteins fresh -- that is, i keep purified,
> > dialyzed stocks of my proteins at -80 prior to applying them to the beads,
> > and thaw them when it is time to bind them to the beads.  one hour of
> > binding shoud be more than adequate before you add whatever extracts you are
> > trying to pull down from.
>
> I have thought of that too.  I am trying to eliminate the time involved
> in eluting the fusion proteins and dialysing them, and then rebinding
> them - roll it all into one - just freeze the initially washed beads.
> Also, some of our constructs don't survive dialysis!!

some proteins are just so stubborn!

> Hence my original question.  Would it be feasible to simply bind our GST
> fusion protein to the GSH beads, wash them, run a minigel to check
> loading level, then freeze the lot in aliquots.  I am trying to find the
> fastest, simplest approach that will be generally applicable to all our
> protiens.

that should be fine--especially if you are doing your pulldowns to probe with
antibodies, and you are not too concerned about trace contamination on the beads
from loading the original bacterial lysates.  i was silver staining the gels
after washing the beads and loading them on SDS-PAGE.  it's amazing how much
contamination the silver staining picks up from the bacterial lysate sticking
nonspecifically to the beads during the initial protein loading. re-loading the
dialysed proteins eliminated that problem for me.  but it was a problem only
because i was silver staining, otherwise i would have done it the way you
propose.

--anton





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