Protocol Online logo
Top : Forum Archives: : Protein and Proteomics

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 ?

-lauranna-

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 sad.gif

-vadakkan-

Im 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

-serglom-

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), Ill try with other blocking solutions (BSA or perfect-block)

-serglom-

I did Westerns with exactly this Cell-Signaling-AB and it worked perfectly with the Amersham ECL detection system and 50 g protein from heart tissue on nitrocellulose membrane after blocking 2 h RT with 5 % low fat milk powder in TTBS. cool.gif

Lea

-Lea Lewin-

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.

-mizzoupharm-

QUOTE (mizzoupharm @ Feb 5 2005, 07:52 AM)
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?

-biolove-