The primary antibody I am using is mouse monoclonal (1:1000) and secondary antibody (1:2500) is goat antimouse IgG-HRP. I am following the standard protocol of Sambrook by adding the recommeded NiCl2. But I am not getting any band. Is there anything that I am missing? My sample is human serum. I have also checked the protein bands after the transfer by Ponceau S. Please suggest me to solve this problem.
DAB=6mg in 9mL of 0.01 M Tris.Cl (pH 7.6), 1mL of .03% NiCl2. And 10uL of H2O2.
Is your loading control staining? If not, then the problem is likely the detection antibody.
Have you titrated the primary antibody, and has it worked in the past?
I have run the same protocol and used Chemiluminescence detection system and I got the signals.
Then it's probably your secondary antibody. Borrow a different one from a colleague and test that (you can re-probe an old membrane if you've stored it iin buffer).
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