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 ?

Hi Lauranna,

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

Vadak

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.
Sincerely,
Sergio

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.

Lea

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.

I've been dealing with p38 from CST for about three years and have finally got it to work for me. It definitely depends on the cell lysate type you are using, primary vs cell line, etc. My lab uses rat primary cultures and run a gel using 40-50ug of protein. Cell Signaling suggests 1:1000 but that doesn't work for me. I use the p38 antibody at 1:100-200 in 1% BSA. I also have to use Pierce's Super Signal Dura West Detection kit. Good luck!

i have been using the phospho kit from cell signaling, works fine. First time i tried it also got non-specific bands, i then stripped the memabrane (0.2M glycine) and it worked fine. Used 1:1000 dilution and methods as stated in their protocols.

Tested the antibody- both phospho and non phospho- with HMVECS stimulated with VEGF-A, loaded 20ul of cell lysate/well, worked well. Also found that i had to bump up the cell/seeding density. If you cant detect anything i would suggest doing an IP