Secondary Reagents

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Related: IgG, streptavidin, biotin, fluorophore, western blot, ELISA

Secondary antibody conjugates are utilized for the indirect detection of a target antigen. They do so by detecting and binding to unconjugated primary antibodies targeted to a specific antigen. This indirect detection method is advantageous as it improves detection sensitivity by promoting signal amplification.

For example, multiple secondary antibodies conjugated to fluorophores will bind to a primary antibody-antigen complex. This interaction results in several fluorophores indirectly binding to the target antigen generating an amplified signal. This allows for the detection of a target of interest, even if the target is low in abundance in a complex biological sample.

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HeLa cells were incubated with (Tubulin+) or without (Tubulin-) mouse anti-tubulin followed by iFluor™ 488 goat anti-mouse IgG conjugate (Green, Left) or Alexa Fluor® 488 goat anti-mouse IgG conjugate (Green, Right), respectively. Cell nuclei were stained with Hoechst 33342 (Blue).

Signal amplification can also be achieved using the labeled streptavidin-biotin (LSAB) method. LSAB exploits biotin’s high affinity to streptavidin as a detection method. Biotinylated secondary antibodies bind to streptavidin conjugates tagged with either an enzyme or fluorophore. Streptavidin conjugates can bind four biotins per molecule in order to amplify the signal. The LSAB method is flexible as it may be integrated into any immunoassay.

Secondary antibody conjugates also provides researchers with an efficient technique for immunolableing. Primary antibodies can be raised to have the same structural constant region but different variable regions to recognize various antigens. A secondary antibody can then be raised to recognize the constant region of these primary antibodies to indirectly detect various antigens. As a result, only a singly secondary antibody requires labeling versus labeling every primary antibody which reduces both time and cost.

AAT Bioquest offers a diverse selection of labels such as fluorophores, phycobiliproteins and enzymes already conjugated to secondary antibodies. Our secondary antibody conjugates are well-suited and optimized for use in various immunoassays such as flow cytometry or fluorescence microscopy.

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