Selecting the right antibody for your application is sometimes like buying a pair of shoes- you may have to try on a few to get the right fit. It turns out that you may also want to walk around the ‘block’—or blocking reagent, in this case, to get the right fit for your antibody. We recently experienced an example of the blocking agent, used to block non-specific binding of antibody to nitrocellulose film slides, creating high background due to cross-reactivity with the antibody probe. This partially defeated the purpose of using a blocking agent….
The situation was this: we were using antibodies to probe for antigens that were spotted down in tissue lysates on the nitrocellulose (what is called a Reverse Phase Protein Array, or RPPA). Interestingly, some antibodies appeared to create a high background on our nitrocellulose compared to other antibodies, using the exact same protocol. We then compared our non-protein-based blocking buffer (Super G) to a casein-
based buffer and the ubiquitous ‘powdered milk’ commonly used in many immunoassays. Over long incubations (12 hours or more) with blocking buffer, the protein-based blockers resulted in a higher background than with shorter incubations, but only with certain antibodies. What we suspected was that some antibodies were cross-reacting with the protein in the blocking agent, and that over longer periods of incubation with the blocker, more of the ‘non-specific’ protein was being bound to the nitrocellulose, and the cross-reactivity of the antibodies was more pronounced.
This observation has raised the question of how ‘non-specific’ bovine milk- or serum-based blocking proteins are from the antibody point of view? If you think about it, cattle raised for food production are immunized, fed hormones and otherwise medically treated in ways that could change their protein and immune profile. So the very antigen that your assay is probing for could in fact be present in the blocking agent, and your specific antibody is now specifically binding to your non-specific blocking agent. Of course you will know in your initial assessment if background is an issue with your antibody of choice. But in the case that you are conducting a longitudinal analysis over time and probing with multiple antibodies, you may want to consider a non-protein based blocking agent.
10 Tips for Selecting the Right Antibody, Genetic & Engineering news.