Western background staining with Primary antibody (Rabbit)

Sharon Waldrop via methods%40net.bio.net (by Sharon.Waldrop from utsouthwestern.edu)
Wed Aug 13 18:16:38 EST 2008

Hello Group.

When I was working with Drosophila embryos, we would preincubate the primary antibody (overnight with rotation at 4 degrees) with embryos that did not have the antigen of interest thus taking care of background (worked great).

Now I am working with cell lines. With my primary antibody to cell surface receptor, I find that if I fix cells that do not have receptor (100 million) with 4%pfa and then wash the cells with PBS a couple of times. I then add 100ul of primary antibody, put on rotator overnight at 4 degrees and bye-bye background on lysed/treated plasma membranes. Westerns look great.

What can I do about an antibody against an intracellular component in the cytoplasm (rabbit, of course)? I am having to use the antibody/5% dry milk/TBST 4 to 5 western run times on Nitrocellulose to get rid of the background before I get clear bands. Please do not suggest PDVF membrane (10X worse). I have also tried different concentrations of antibody (no difference).

It is not the secondary, already looked at that as the cause of problem (use it with the extracellular - works fine). Pulling my hair out as well as wasting time and reagents. Any suggestions? Thanks.

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