western blot problem - antibodies can't detect the protein on the membrane (Feb/09/2006 )
I'm running 10% SDS-PAGE followed by transfer to nitrocellulose membrane. After staining with ponceau clear bands are shown (at the right place). The problem is that the antibodies I'm using don't seem to detect the protein on the membrane. The antibodies work in other systems. I've tried different dilutions of the secondary antibody (I can't change the concentration of the first antibody), and still nothing. Not even background...
Can anyone help?
what are other systems?
be careful, some antibodies detect the antigen in the native conformation but not denatured.
do you have a control with purified antigen?
I've used both the primery antibody and secondary antibody under same conditions before , for the same protein (IgM) from another type of cells, and it worked perfectly. the only difference now is the source of the the IgM, I use a different type of cells now. I use IgM the old cells as positive control, but I don't get any signal even for that. I have no reason to believe the primery antibody is not working properly...
I think you have every reason to think that something happened to the AB "I use IgM the old cells as positive control, but I don't get any signal even for that".