MAPK P38 - troubles with western blot (Oct/15/2004 )
I m having troubles with my western blots.....
I try to reveal the MAPK P38 (phosphorylated) with cell signaling antibodies but no band appears. P38 antibody is at 1/1000.
My procedures are OK with other antibodies. Did someone have troubles with this antibody ?? How many micrograms of protein (or cell lysat) do you load per lane ?
I also had the same trouble with cell signalling antibody.
Did you repeat the experiment?
Let me know if you have found out a way
I´m just doing a western for p-p38, but my problem is different. I have too many bands in the membrane after revelation. So I have bought an inhibitor of p38 phosphorylation in order to localize the correct band. The antibody recognize Tyr 182 of the protein and it is at 1:200 (santa-cruz recomendation). I will post my results when the blot have finished.
I only see inespecificity bands and after 20 minutes of exposure... it could be the blocking solution (5% non-fat dry milk in TBS-T), I´ll try with other blocking solutions (BSA or perfect-block)
I did Westerns with exactly this Cell-Signaling-AB and it worked perfectly with the Amersham ECL detection system and 50 µg protein from heart tissue on nitrocellulose membrane after blocking 2 h RT with 5 % low fat milk powder in TTBS.
Your not the only person having troubles with mapk p38. Our post-grad and a few other graduate students here are also have a hell of a time with it.
which company is your antibody against p38 MAPK from?