Utilizing two or more independent antibodies for the testing of the same protein target can be a useful tool to determine the specificity of the antibody. Ideally, two or more antibodies which are used can identify the non-overlapping epitopes of an antigen. By comparing the results of the antibodies which recognize the independent regions of the same target protein, the specificity of these antibodies are ensured and these antibodies are suitable for the detection of the target protein.

The applications of independent antibody validation are carried out in western blot, IHC arrays, immune-fluorescence in multiple cell lines and tissue samples. Meanwhile, immune-precipitation, flow cytometry, and other applications are performed.

The following example demonstrates how we use independent antibodies to verify the specificity.

Here, three independent NF-kappaB p65 polyclonal antibodies (ab4, ab5, ab6) are tested in the lysates extracted from 293 cells, mouse brain tissues and rat brain tissues. The immunogen of the three antibodies were derived from different epitopes of the human NF-kappaB p65 protein. Differences in species reactivity and mass spectrometry result suggest that these antibodies have adequately different binding profiles to their target, therefore it is appropriate to verify the specificity of these antibodies. 

As expected, the band of NF-kappaB p65 is prominent. Additionally, ab4 and ab5 show the same pattern of p65 enrichment in rat brain tissues and 293 cells, further demonstrating the specificity of the antibodies.

Since prominent band is detected by ab4 in all the three samples, we choose ab4 as the optimal NF-kappaB p65 antibody. Therefore, we have the corresponding product known as NF-kappaB p65 Antibody, with the cat# of AF5006.

Three antibodies showed good correlation in detecting human, mouse and rat p65 proteins

Affinity antibodies that have been verified using independent antibodies are indicated with an ‘IAV’ symbol in search results and on relevant product pages.