Hi

I dont think there is any problem with your antibody if your antigens are both from the same source( human or mouse)

1) if you are checking in-vitro interaction make sure your antigen is not degraded. better to load your antigen and make sure you get the proper band in expected size.

2) check the antigen source of preparation by the company, sometimes post-translation modifications are involved in binding.

3) check the buffer composition of your antigen

Hi Maryam, are you working with polys or monos.?

Have they been affinity purified using an antigen column or not?

Antibodies that are not affinity purified using small antigen columns are worthless.

dear Steingrimur Stefansson

thank you for your response,I use anti-vitamin D sheep monoclonal Ab, it was purified using size exclusion chromatography.

Hi Maryam, I don't think there is such a thing as a sheep monoclonal antibody.

dear Steingrimur Stefansson

Bioventix company(England) produce sheep monoclonal antibody .