problem with western blot - (Jan/17/2007 )

i have a problem with my western blot (ECL): i can see a lot of proteins in "negativ"..like a goust and i can not see a specific bend at all.
i block the membrane 1h at RT or 4 overnight with skim milk (pbs tween 0.1%)
primery ab 1:10000 (in the same blocker) then wash 4 times 10 min with pbs tween 0.1%
second ab 1:100000 (gout anti rabbit HRP) and wash 4 times 10 min with pbs tween 0.1%

i tried to make dillutions of my proteins samples but still i can not see my specific protein that the ab should recognise (only the black sheddow of the membrane).
what do you think i should do??

thanks
vit.

It seems the primary and secondary antibodies are being diluted a lot. R u sure abt the antibody conc. ? And r u sure abt the specificity of the antibodies. May b try a different aliquot of the antibody.

next time try to use as much of the protien as possible just to have as a positive control. Does ur marker transfer properly?

you see it in negative. so you have a strong background?
are you trying to detect biotinylated or phosphorylated proteins ?

I found on the Abcam website:

White bands on a black blot (negative of expected blot)
Too much primary and/or too much secondary antibody.
Dilute the antibodies more.

http://www.abcam.com/ps/pdf/protocols/abca...ing_tips_WB.pdf

here are more tips for doing western blots

Either your protein or antibody is in excess.