Phospho-Elk1 specific Antibody...which band?? - (Feb/18/2005 )

Hello

I´m working with a Phospho-Elk1 (Ser383) specific Antibody (Santa Cruz) but the detection of the right band seems to be a hard piece of work since there are some bands at the expected size. I already tried the appropriate blocking peptide without any success.
Is nuclear fractioning absolutely necessary for proper detection of pElk1 on Western Blot? (HeLa cells). Somebody got a good protocol?
Could it also be possible that the phosphorylated protein runs even further than the unphosphorylated one? Is it possible at all that you can see a migration difference between pElk1 and Elk1 in Western Blot?

Hope somebody can help me to identify the right band.

-hanze-

QUOTE (hanze @ Feb 18 2005, 12:02 PM)

Hi,
I just came across your question for the P-Elk1 blot. I'm doing a similar blot on protein extracted from endothelial cells-i loaded 50ug protein on 10% gel-blocked with 5% milk and incubated the membrane with 1:200 ilution of teh antibody in 5% BSA- and still saw no bands at all...I was wondering if you coul;d pass on the conditions that you used for the blot and also lemme know if you got to resolve your problem!
Thanks

-ruchi-

Hi,
#1. Do you know for sure that the antibody works? Santa Cruz antibodies are often CRAP!
Has anyone published anything using this antibody?
#2. If someone has published something use what they did as a positive control.
#3. Treat Hela extract with a phosphatase (lamda phosphatase works well). This should work
as a negative control because you should lose your band in this lane.

-mikew-