I am trying to see if the antigen is on the nuclear surface or inside the nucleus, but the antibody is giving non-specific background, even in the cells which are not supposed to have this antigen. It is only after blocking with "super-block" for about 12-16 hrs (done empirically) that the background reduces to a minimal level, but not completely.
If your antibody has a high background, it's not because from IgM. You may try a different antibody. Good luck!
please visit this website
http://www.protocol-online.org/cgi-bin/prot/view_cache.cgi?ID=170
Thanks Hongjuan Cui. This is unfortunately the only antibody available for my use. I have tried to play with various blocking reagents, incubation times, washings, cell fixation reagents etc. Isotype control shows no signal at all.
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