Blocking

A blocking agent is used after the coating step. This agent is an irrelevant protein that does not interfere with the experiment itself. It covers all unsaturated surface-binding sites in the microplate wells, thus preventing nonspecific binding of proteins to the microplate during subsequent steps in the assay. Nonspecific binding may result in false positives. An optimal blocking buffer maximises the signal-to-noise ratio and it is important that it does not interact with the antibodies or the target molecule.

The blocking agent may be a protein or a detergent (or a broth). Common blocking agents are Tween 20 (a non-ionic detergent), non-fat dry milk, gelatin, and bovine serum albumin (BSA).