Western background staining with Primary antibody (Rabbit)

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.

In article <[Only registered users see links. ].net> , "Sharon Waldrop" <[Only registered users see links. ]> wrote:

The best (but more laborous) thing to do:

Extract cytosolic proteins from the cells (hypotonic lysis/dounce),
spin in ultracentrifuge to remove all insolubles, concentrate to
at least 5 mg/ml protein, dialize *extensively* (to remove small
molecules) against secondary amine-free buffer, couple to
1-3 ml of an immobilized support (1:1 mixture of Bio-Rad's AffiGel 10
and 15 would work best; 5-10 mg/ml of activated gel), pack
into a small column, then run your antibodies over it (reload
flow through to be sure). Zero background guaranteed.

We just did it here with some plant material.

DK

your antibody over this Purify

Am 14.08.2008, 11:01 Uhr, schrieb DK <[Only registered users see links. ]>:


However, you may get away with just incubating the cytosol with a membrane
rather than doing a coupling reaction. That should be a lot quicker and
cheaper. In this case however I would prefer PVDF over NC because of the
stronger protein binding.