Anti-Involucrin antibody (ab53112)

Thank you for calling about the issue you are having with the involucrin antibody ab53112. The amino acid sequence of the recombinant fragment that you are using as a positive control, ab73744, overlaps the sequence of the immunogen at 11 of 14 amino acids, so the antibody should be capable of detecting that fragment, assuming that at least some of the epitopes it recognizes are present on the fragment.

I suspect the antibody is defective, if you are confident that the capillary western protocol and other materials are known to be effective. Are you confident that the secondary antibody and detection reagents are not at fault?

For membrane isolation protocol, I recommend the protocol at the following link:

https://www.abcam.com/index.html?pageconfig=resource&rid=11473

For controls for the antibodies you listed, I recommend the following:

ab15098 - Claudin - HeLa cell lysate ab29545
ab15148 - E-Cadherin - MCF7 cell lysate ab3871
ab64482 - Occludin - Use a human kidney lysate such as ab30203.

I look forward to your reply. I will be happy to suggest a replacement for ab53112, if you are confident that there is no other explanation for the lack of signal for involucrin.

Thank you for your reply with further information. The image suggests the higher band may be the correct band as it is only present in the first four samples as expected. You previously describe a higher band at 120 kDa and I assuming this is that band. We have another involucrin antibody ab17105, which detects a specific band at 170 kDa. Furthermore I have reviewed the literature and a a 120-170 kDa protein is reported in some cell lines. Therefore this higher band is likely to be the target protein. Here are some references that may be of use. I hope this information helps.