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once more p38



Dear Yair,

yes, I used anti-phospho p38 for immunoprecipitation, because it was in the p38 MAP kinase-assay kit from NEB.

No, I do not know whether the anti-phospho ATF-2 antibody is working. Do you know a positiv control to test this?

For cell lysis I used RIPA-buffer (containing NP-40 and sodium deoxycholate) and dilute the sample in NEB-lysis buffer (containing Triton X-100) prior to immunoprecipitation.
 

Dear Jim,

may be I will try the agarose-conjugated anti-p38 antibody from Santa Cruz. Which antibody do you use? sc-535 AG or sc-728 AG???

Do you perform a pre-clearing step before immunoprecipitation? I found, that pre-clearing with Protein A-agarose beads "precipitated" the p38!!!

Then I tried immunoprecipitations with Dynabeads (M-280 sheep anti-rabbit IgG coated) and anti-p38 antibody (sc-7149), but it does not work very well. Most of the p38 protein stays in the lysate.
 

Dear Marianne,

thank you for the p38 kinase-assay protocol!

One further question: Which kind of sepharose beads do you use for antibody coupling?
 

Thank you all for help.
I wish you a happy week-end and I hope I will hear from you (perhaps) monday.

Best regards,
Brigitte