The 2 monoclonal antibodies may recognize different regions of the target protein. There could be enough difference in conformation, steric hindrance due to glycosylation or other attachments, or amino acid sequence that the rabbit mAb failed in 2 of the cell lines.

Ellen A G Chernoff

Thank you so much for answering!!

I understood that.

I have another question. Is it worth investigating the details of the difference? And why?

I want to hear your opinion.

If you know, would you tell me how to check the region that these antibodies recognize etc?

In best case scenario, you could simply ask the manufacturer whether the epitopes of the antibodies are known.

Experimentally, it can be tested using different truncation mutants (for example with N-terminal tag to see them all as control), but would of course require some effort to generate the mutants and also some luck to narrow down the epitope region quickly. Otherwise just search for "epitope mapping methods" to get more ideas.

Whether it is worth doing may depend on overall focus of your research. Given that 2 of analyzed cell lines seem to express it differently, could reveal an interesting phenomenon.