blocking for western blot

Nick Theodorakis nicholas_theodorakis at urmc.rochester.edu
Sat Apr 22 13:51:55 EST 2000


In article <v04011702b524b6ab1c96@[144.92.64.174]>,
  mlsulliv at facstaff.wisc.edu ("Michael L. Sullivan") wrote:
> A collegue once did a sort of study for what blocking agents worked best
> for her situation.  She found (for her Ab) that milk was horrible (very low
> background, but low signal too) and that 3% BSA worked the best (high
> signal, low background).  I suspect that what might be the best blocking
> agent might depend on lots of things, like what the membrane is like, what
> your samples are like, and the nature of your primary and secondary Ab, so
> you might just have to try lots of stuff emperically.  I think some of the
> common blocking agents, are BSA, dry milk, "special" commercial variations
> on dry milk, and tween-20.
>
> Hope this is useful.
>
> Mike
>

We have found through trial and error that 5% milk in TBS/Tween works the
best for _most_ of our Abs, for some reason that 1% BSA works better for
Promega's anti phospho-erk Ab, in our hands. Which is what they actually
recommend, but who would'a thought they'd be right? ;-)

(We're using Amersham's ECL as detection, BTW)

Nick

--
_______________________________________________
Nick Theodorakis
nicholas_theodorakis at urmc.rochester.edu


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