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A secondary antibody is an antibody, much like any other; its nomenclature stems from its place in the application or experimental setup. The secondary antibody is used to detect the antibody specific for the protein of interest (POI) – the primary antibody. Predominantly the secondary antibody is conjugated to the detection reagent such as fluorophore or reporter enzyme but could be an unconjugated “bridging” antibody used in an ELISA. Of course, a secondary antibody could be an antibody that isn’t specific to the primary antibody but could be a tertiary antibody in the experimental setup.
A protein of interest (antigen) is specifically recognized by a primary antibody, which may be directly conjugated (direct detection, Fig 1.A); or the primary may itself be detected by a conjugated secondary antibody (indirect detection, Fig 1.B), allowing visualization of the protein/analyte.
The indirect method using a secondary antibody offers many advantages over the direct method.
Learn more about choosing your secondary antibody here
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|Multiple labeling using Secondary Antibodies||Whole IgG affinity-purified antibodies|
|Direct and Indirect Western blotting||Build dye panels – Spectral Viewer|
|Choosing your Secondary Antibody||Secondary Antibodies for VHH discovery|