Simple anti-His WB not working - (Nov/07/2007 )

I tried to detect some His-tagged proteins today by WB. As primary Ab I used mouse anti-His Ab (1:1000, Dianova) und AP-conjugated goat anti-mouse IgG Ab (1:5000) as secondary Ab. When I developed the blot in 10 ml AP buffer and BCIP and NBT really nothing happened. The positive control didn`t show a band and there was also no sign of unspecific binding. Normally, due to incomplete washing, I see at least some colour f.ex. in the corners of the blot.
I checked the protein transfer from gel to membrane by Ponceau staining and the positive control was clearly there.
I came down to 3 possible problems: one of the antibodies or the AP buffer/NBT/BCIP.
What is your opinion?

Thanks, Isa

I would try to make everything fresh like the buffers etc. Then you could ad more first antibody (like 1:200, 1:500), try a dilution series with just positive control and mock. Then you should get some result at one point. Also check if your second antibody is working, perhaps with another first antibody or put the antibody on the membrane and develop. How long do you let the first ab incubate on the membrane? You should let it sit at least for 2 hours, better over night.

Hope,this helps