“Know Thyself – and Thy Primary Antibody!”
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.