The purpose of the blocking step in an assay is to improve assay sensitivity by reducing background interference. However, unforeseen cross-reaction of detection reagents with blocking buffers is itself a cause of high background and low signal-to-noise ratioses in assay systems. Because individual blocking buffers are not compatible with every system, a variety of blockers in both Tris-buffered saline (TBS) and phosphate-buffered saline (PBS) are available. The best blocking buffer for a specific experiment will bind to all potential sites of nonspecific interaction, eliminating background without altering or obscuring the epitope for antibody binding.

To optimize the blocking step for a particular immunoassay, empirical testing is essential. Using inadequate amounts of blocker will result in excessive background. Using an excessive blocker concentration can mask antibody-antigen interactions or inhibit the marker enzyme. For best results when developing a new immunoassay, test several different blocking agents for the highest signal-to-noise ratio in the assay. There is no single blocking agent that is ideal for every occasion because many factors can influence nonspecific binding, including various protein interactions unique to a specific assay system.

Features and Benefits:
‧ Popular – nonfat milk has been used for many years in a variety of protein methods, although it is not recommended for avidin-based techniques because it contains some endogenous biotin
‧ Convenient – supplied as a ready-to-use 1X TBS solution; can be diluted as needed
‧ Easy to use – formulated with anti-foaming agent and thimerosal-free preservative
‧ Flexible – may be used for multiple applications, including as a diluent for antibody