Figure 1. Linearity comparison of COX IV rabbit monoclonal primary antibody (P/N 926-42214) to β-Actin rabbit monoclonal (P/N 926-42210). Primary antibodies were compared by Western blot and detected with IRDye 800CW Goat anti-Rabbit (P/N 926-32211). The COX IV antibody can be used as a mitochondrial loading control and a loading control for normalizing low expressing target proteins. This COX IV primary antibody remains linear with increasing concentrations of lysate, making it ideal for normalization.
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.
Okay, so you’ve done your experiments, run your sample on a gel, and transferred the proteins to a membrane. Now, you need to see if you can detect the protein, what happened to it, how much is there, etc.
After you block (remember we talked about how important the right blocker is), you will probe with a primary antibody (that is, an antibody produced to detect a specific antigen) to see your molecule of interest. Now, primary antibodies can be produced in a wide variety of species such as mouse, rabbit, goat, chicken, rat, guinea pig, human, etc. There are lots of suppliers of antibodies out there, so it is important to realize primary antibodies for the same antigen can perform very differently. It may be necessary to test multiple primary antibodies for the best performance in your Western blot system.
In the images below, you can see how different primary antibodies to the same target may react. Serial dilutions of NIH/3T3 lysate were probed with Akt monoclonal primary antibodies from three different vendors. All blots were blocked with 5% skim milk and detected with HRP-conjugated Goat Anti-Mouse and SuperSignal® West Dura chemiluminescent substrate. Western blots were imaged on the Odyssey Fc Chemi channel for 2 minutes, shown with normalized image display settings. You can see that the primary antibodies varied quite a bit. The number and intensity of bands you can detect and the amount of non-specific binding that occurs are definitely different for each one.
So, take a cue from ancient Greece and get to know your Primary Antibody by doing some testing and optimization.