Chemiluminescent Western Blots – FAQs about Primary and Secondary Antibodies

We’ve discussed some hints on how there can be considerable difference in primary antibodies – so “Know thy Primary Antibody.

In addition, we’ve received some questions that are frequently asked – hence called frequently asked questions or FAQs – about primary and secondary antibodies when doing chemiluminescent Western blots. So here they are. I am sure there are more. . .so send them our way by commenting on this post!

Why is the signal missing in the middle of the bands?
Too much secondary antibody on the membrane results in consumption of all the substrate in that area. Without substrate, there is no chemiluminescent signal and a white spot appears in the center of the band. Try different dilutions of the primary and secondary antibodies to find what gives the best results, or try changing the substrate.

Does it matter where I purchased the HRP-conjugated secondary antibodies?
The reactivity of secondary antibodies ranges widely between vendors. As well, the ratio of HRP enzyme to antibody varies, and may affect the detection of the target. If the secondary antibodies from one vendor are not working, trying antibodies from other vendors may be helpful.

Should the HRP-conjugated secondary antibodies be highly cross-adsorbed?
Although highly cross-adsorbed antibodies are essential for two-channel, multiplex detection, it is not always necessary with chemiluminescent blotting for a single target.

What questions do you have?

Optimizing Chemiluminescent Western blots Technical Note might be a good place to start to get some of those questions answered. And remember you save time and money by going digital with the Odyssey® Fc Chemiluminescent and Infrared Fluorescent Imaging System!

Happy Blotting!