So we’ve talked about choosing primary antibodies , and you have come to know the ones you are using very well. We have also walked through some frequently asked questions about primary and secondary antibodies for chemi Westerns. The next step in Western blotting detection (after washing the blot, of course) is to probe with the secondary antibody (also known as secondaries, secondary conjugates, antibody conjugates).
As you can see in the images below, the reactivity of secondary antibodies ranges widely between vendors, even within the same species and especially between host species. The ratio of HRP enzyme to antibody varies and may affect the detection of the target. Try secondary antibodies from several vendors to find the ones that give the most satisfying data.
Serial dilutions of mouse or rabbit IgG were spotted onto nitrocellulose (2500 pg to 0.3 pg) and probed with HRP-conjugated secondary antibodies from various vendors. Blots were detected with SuperSignal® West Dura chemi substrate (Thermo Scientific) and exposed to film for 15 seconds.
Another one of those Notes: When evaluating the performance of the primary and secondary antibodies, try different blocking buffers (yes, we had a post on that too – 8-Dec-11 - The Best Offense is a Good Blocker), as the choice of blocker can affect the antibodies’ performance.
For optimal results do not dilute the HRP-conjugated secondary antibodies with blocking buffer containing sodium azide as a preservative (e.g., Odyssey® Blocking Buffer), as it will inhibit peroxidase activity and result in less light production.
Don’t want to use film?? (ugh! all that mess and expense!) Then, you might be interested in the Odyssey Fc Chemiluminescent and IR Fluorescent Imaging System.
For more optimizing tips for chemiluminescent Western blotting, see our Technical Note. And, of course, check in here again for more tips and hints on various applications that can be performed on the Odyssey and Pearl imagers.