M2 antibody background in Xenopus cell extracts - (Dec/13/2013 )

Hello fellow scientists :)
I am working with Xenopus laevis oocytes and I've been experiencing very strong background bands on my PVDF membranes when i try to detect my Flag-tagged proteins of interest using the M2 antibody from Sigma-Aldrich. The size of these background proteins is 40-45 kDa, and the signals are often much stronger in intensity than the signals of the specific bands.
I tried 2h blocking in 5% skimmed milk in PBS with 0.05% Tween, followed by 2h incubation in M2 in PBS+0.05% Tween (1:30 000) and 1h of GAM-PO in PBS+0.05% Tween (1:10 000)
as well as the overnight incubation in M2 in PBS+0.05% Tween (1:30 000) without blocking. both methods seem to give very similar results. 

So my question is: has anyone experienced similar problems while working with xenopus oocyte extracts, and does anyone have any idea how to solve this?

Try diluting both antibodies in your blocking solution.  Try increasing the number of washes between antibodies.

Are  you doing an IP by any chance?  If so - the bands are likely to be heavy chain of IgG and can be eliminated by using a primary antibody from a different species to the one you did your IP with.

oh i forgot to note that i actually also put some skimmed milk powder in PBST in which i dilute my antibodies in. usual washing consists of three washing steps with PBST (10 min each) on a shaker at rt.
i'm not doing IP. i'm analyzing the distribution of my proteins of interest in a sucrose gradient after ultracentrifugation. each fraction is precipitated by Wessel and Flügge (1984), ran on 10% acrylamide gels and transferred on a pvdf membrane.
the background signals are noticeable only in the last 5-6 fractions (25-35% sucrose)

you may be seeing an artifact (presumably keratins from dust) caused by aging reducing agent in the sds-page sample buffer. you can determine this by running blank samples along with your regular samples.

we often run gels for blotting without reducing agent to avoid this (you can also use a fresh lot of reducing agent), but you will need to determine if you can do this with your own protein of interest.

Thanks, but I already checked that and everything was fine. Also, all the solutions used in the process were freshly prepared.
This most probably is a cytoplasmic protein of 40-45 kDa that the M2 antibody (anti-flag) recognizes, i can see it only in the five last fractions of the gradient. i even tried blasting the flag-tag against all known xenopus proteins, but got no relevant hits :/

(oops, missed that part about the last 5 fractions)

a couple of things:

first, determine if it's the primary or secondary antibody which is binding to the 40-45 kDa protein

then try more dilute solutions of the offending antibody

add additional blocking agent(s) to the offending (or both) antibody solution(s) (eg-bsa, normal serum from the species in which the secondary antibody is produced). this may pre-absorb the portion of the antibody which binds to the 40-45 kDa protein.

the primary antibody is the problem since the secondary antibody was used in the combination with other antibodies (like for beta-actin) and no background was visible. at this point, i will just abandon the overnight incubation at 4°C and do the 2h incubation at 37°C, with which the background signal is still there, but less intense that with the overnight incubation at 4°C.
Thank you all for your help! :)

in case anyone who uses Xenopus laevis as their model organism and works with M2 antibody from Sigma has noticed these background signals, please contact me :)