How to remove RNase from immunoprecipitation?

Nick Theodorakis nicholas_theodorakis at urmc.rochester.edu
Thu Sep 14 16:04:49 EST 2000


In article <7opiGDf98QgB-pn2-kovHnGUflsZI at rnaworld.bio.ukans.edu>,
  PGegen at UKans.nolospamare.edu (Dr. Peter Gegenheimer) wrote:
> OK, I've had it! We are trying to do a "simple" immunoprecipitation in which
> the unbound fraction will be assayed for RNA processing activity. We find that
> protein A-agarose beads have little non-specific RNase activity. After adding
> serum (immune or non-immune),  we wash the beads up to 10 times until the wash
> supernatant has no detectable RNase.
>
> However, when we do the actual IP by incubating these beads with the
> experimental enzyme fraction, the unbound supernatant has much more
> non-specific RNase activity than did the enzyme fraction before IP! This
> non-specific RNA breakdown isn't inhibited by RNase inhibitor or carrier RNA.
>
> Is there some simple step we've overlooked? Any suggestions, however, wild,
> will be welcome!
>

Could your antibody have removed an endogenous RNase inhibitor present in
the extract?

Nick


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


Sent via Deja.com http://www.deja.com/
Before you buy.






More information about the Methods mailing list