Why does my functional blocking antibody only work at lowest conc? - (Aug/14/2014 )

Hi,

I am trying to do some antibody blocking experiments for the first time. I pre-incubated my cells for 1h with 0.1ug/ml, 5ug/ml and 10ug/ml of functional antibody (sodium azide removed). I included isotype controls. I see fantastic blocking of signalling at the lowest concentration, which made me very happy. But I see no change with the higher two concentrations. I find this strange. I have read that some antibodies can cause receptor activation when binding, but I don't think this explains it, as then I would get higher signalling with the higher concentrations, whereas I get no response at all.

Any advice to help me understand this would be greatly appreciated!

Thanks

No change meaning no change from the unblocked or no change relative to the lower concentration?

If it is relative to the lower concentration, then it is possible that you are at a saturating level already.

Thanks for the reply. No change in higher doses relative to unblocked, i.e.:

NT= 100%

0.1ug/ml= 8%

1ug/ml= 100%

10ug/ml= 100%

Those concentrations might be causing the antibody to precipitate or aggregate, especially if there are other antibodies in the medium (e.g. in serum).

Oh that's an interesting thought, thanks. Does this mean I should be doing these experiments in lower/no serum conditions? Sorry if this is an obvious thing I should have been doing anyway- completely new technique for me and hard to find info online, as mostly WB protocols come up with the search terms.

No idea sorry, but it would be relatively simple to test...