blocking for western blot

Andrew J Ippolito aji97 at acsu.buffalo.edu
Tue Apr 25 18:36:42 EST 2000


We actually use PBST now rather than TBST.  PBS is readily available from
the tissue culture we also do in our lab, and is apparently cheaper.  I have
noticed no differences in the quality of the results.  For blocking we use
5% powdered milk (also Carnation brand) either 1 hour at room temp or
overnight in the coldroom (it's a nice place to stop for the day if you
began late).  We notice that you must completely dissolve the milk before
use, which takes 1-2 hours (make it while the initial gel is running).  I
personally freeze aliquots to save time.  We do all primary and secondary
incubations at room temperature for 1 hour on a rocker, and do NOT add milk
to them; the primary can thus be used indefinitely (or at least it lasts
longer than the 3 weeks or so the ones with milk do).  We find this does not
significantly contribute to background levels.  Washes are either 3x10' or
4x5' PBST.

Question:
>so everything  is still "fresh".  Even a night in the cold room probably
doesn't do much
> good for your samples.

I don't understand how a transferred protein on a nitrocellulose blot may
suffer from sitting overnight in a cold room.


--Andrew Ippolito
RPCI
Buffalo NY







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