high non-specific binding on monocytes by flow - (Feb/29/2008 )

I'm using flow to identify a membrane protein (let's call it P1) and am having lots of problems with non-specific binding in both samples with the primary antibody and the negative control. I hope someone can help. Here are the details.

I am trying to identify a human protein (P1) using a rabbit anti-human P1 as a primary antibody. The secondary antibody is a goat anti-rabbit IgG - FITC. The FcR's on macrophages are blocked with goat serum. I tried a serial dilution of the primary antibody and also had a negative control with rabbit IgG. Upon doing flow cytometry, the tube without any antibody did not have any FITC signal, however, all other tubes, including the ones with primary and the rabbit IgG lit up!!

I am puzzled as to why this happened. The fact that the 0 primary Ab tube didn't light up suggests it is probably not the lack of FCR blocking with goat serum. It appears it might be something common to both the rabbit IgG and rabbit anti-human P1. Does this make sense? The IgG should not be binding, should it?

Does anyone have any ideas why there is so much non-specific binding of the secondary in both the test and rabbit IgG samples? What else can I do? I've read that macrophages are notorious for this. Any ideas are much appreciated.

Thanks

hi

1. Sreum is not the best way to block the Fc receptor i would advise using Fc Block (form ebioscince or any other).
2. Try to use F(ab)'2 secondary antibodies (i.e. the antibody without the Fc) or conjugated antibodies (i can highly recommend Biolegend - they are very good and CHEAP !!!).
3. do all the steps in PBS/1% BSA 0.05% NaN3 (azid). make sure the use a BSA suitable for staining.

All the best,

L.