How to remove RNase from immunoprecipitation?

Dr. Peter Gegenheimer PGegen at UKans.nolospamare.edu
Thu Sep 14 15:30:46 EST 2000


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!

Thanks,

o----------------------------------------------------------------------o
| Dr. Peter Gegenheimer       | Vox: 785-864-3939  FAX: 785-864-5321   |
| Department of               |   PGegen at UKans.nospam.edu              |
|   Molecular Biosciences     |   http://rnaworld.bio.ukans.edu/       |
| University of Kansas        |"When you have excluded the impossible, |
| 2045 Haworth Hall           |  whatever remains, however improbable, |
| Lawrence  KS  66045-2106    |  must be the truth."      S. Holmes    |
o_____________________________|________________________________________o






More information about the Methods mailing list