I am doing work on prostate cancer cell line - LNCaP - and using PSA antibody to show androgen deprivation. We currently have 3 in our lab, none of which are giving us a signal for western blots. They have worked previously for someone else in the lab - all that being said not very well. We have the Santa Cruz (A67-B/E3): sc-7316 and 2 Abcam 1 mouse monoclonal (A67-B/E3 ab2218) and 1 rabbit monoclonal (EP1588Y ab76113). All are stored at 4C as stated on data sheet and have been used as concentrated as 1.100 with no signal. I even tried a native blot which again no signal was observed. Any help would be greatly appreciated.
A lack of signal on an immunoblot could be due to a number of reasons. Therefore, a careful analysis may be needed. Inclusion of a recombinant PSA protein or a positive control from a previous sample in the blot could rule out antibody-related issues. The antibody that you listed from Santa Cruz has worked in our hands.
I would agree and propose to revise your protocol. If antibodies produced a reliable signal in the past, the most probable source of uncertainty may be the experimentator self.
Thus, you should verify different points in your procedure, such as protein conc. in your sample, SDS-PAGE, efficacy of blot/transfer, antibody conc., buffers, dyes etc... point by point
Agree with previous answers. Is the positive control working? Are you sure the protein quality and quantity in your samples proper? Revise your procedure for other errors.
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