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

Anton Tutter atutter at aim.salk.edu
Sat Mar 4 18:40:50 EST 2000


> ð We attach GST-pusion proteins to GSH-agarose, and need to keep the beads
> ð for a long time for a frenzy of pull-downs we are doing.  We find they
> ð get bacterial contamination or lose activity after a month or so at
> ð 4degC.

 
> Some beads -- I think including agarose -- are shipped in 20% EtOH. Unless
> this would kill the glutathione, you could store the beads at 4deg in 20%
> EtOH.
> 

i wouldn't do that if i were you!!!  i think peter misunderstood the
questions -- the beads are to be stored long-term *after* the GST-fusion
proteins are bound to the beads.  20% ethanol may denature the proteins.
what i would do is rapid-freeze the bead/protein complexes in liquid
nitrogen, making sure there was 20% glycerol in the final buffer before
storing. then store at -70 or colder.  the proteins should last a good long
time that way without losing any activity.  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.

what's the nature of your GST "bait" proteins?  i routinely use
transcription factor activation domains as bait and they generally are
resistant to at least two or three freeze/thaw cycles, provided there is 10%
glycerol or more (also slight detergent sometimes helps maintain solubility,
like 0.1% NP-40), and i freeze/thaw them as rapidly as possible (nitrogen
freeze, room temp water bath thaw).

--anton

tutter at salk.edu





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