[Yeast] Help with IP and RNase A treatment
(by skervestin from yahoo.fr)
Thu Aug 30 11:03:05 EST 2012
I'm doing co-Immunoprecipitation experiments in yeast (s. cerevisae) and I think the interaction I see between my two favorite proteins is bridge by mRNA (because both are able to bind RNA).
I'm looking for a protocol to perform RNAse treatment of my extracts before IP.
First I plan to go with RNAse A but someone advices me to not used it because it could not degrade poly(A)tail protected by the poly(A) binding protein and mRNA in the closed loop conformation. I'm very surprised because RNase A seems to be communly used for this application.
I have several questions :
- does anybody already have trouble degrading mRNA with RNAse A?
- Could you recommend another RNAse (micrococcal nuclease may be?)?
- If RNAse A is fine, does anybody have a protocole with the quantity I should use? My IP experiments are performed in a volume of 1ml with yeast extract at 2 mg/ml (assayed in bradford) in buffer TrisHcl pH 7.4 10 mM, MgCl2 30 mM, NaCl 100 mM, Triton 0.01%.
Thank you very much for your help
13 rue Pierre et Marie Curie
Stephanie.Kervestin from gmail.com/Stephanie.Kervestin from ibpc.fr
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