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Antibody condition optimization includes antibody selection, assay condition optimization, and signal optimization. Primary antibodies that directly bind the substrate of interest can be monoclonal or polyclonal. Monoclonal antibodies to a specific region of a substrate are identical and recognize one site on the substrate. They are produced in mice, are more specific, more consistent between commercial batches, and less cross-reactive with other substrates than polyclonal antibodies. Polyclonal antibodies are mixed populations of antibodies that recognize different sites on a substrate. Despite increased potential cross-reactivity and variation between commercial batches, they are more stable, more able to detect a substrate regardless of its conformation, and they can be generated in several animals. For each antibody and each assay the appropriate diluent for the antibody, antibody concentration, incubation time for binding, and assay blocking solution are determined. The blocking solution reduces non-specific binding by the antibody. Signal optimization ensures that enough labeled antibody binds the substrate for visualization or assay quantification. This involves selecting a direct or indirect detection method, the best label for detection, and an antibody with several labels or one.
(Credit: Brooke Anderson-White)