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What Are Secondary Antibodies?

Secondary antibodies bind to the immunoglobulin (Ig) domain of the primary antibody and are species and isotype specific.

Secondary antibodies are useful tools that can help increase the effectiveness of a variety of experimental methods including Western blot, immunohistochemistry (IHC), flow cytometry, and ELISA. They are generated by immunizing host animals with antibodies from a different species. The antibodies that are generated recognize and bind to the immunoglobulin (Ig) domain of the immunizing primary antibody and assist in purifying, sorting, and detecting target antigens. Secondary antibodies are typically conjugated to a label. The downstream application will determine the conjugation state and choice of label of the secondary antibody. 

Selecting Secondary Antibodies

Secondary antibodies are selected based on several different factors. Since the secondary antibody needs to recognize and bind to the primary antibody, the secondary antibody needs to be specific for the primary antibody’s host species and isotype (e.g., IgG or IgM). Additionally, the secondary antibody should be conjugated with the most appropriate label for the application it is being used in. For example, Western blot and IHC can use secondary antibodies conjugated to either chromogenic or fluorescent molecules, while flow cytometry generally uses fluorescently labeled secondary antibodies.

Bio-Techne primary and secondary antibodies were used to detect CD32/PECAM-1 in the bEND.3 mouse endothelioma cell line by WB.
Detection of CD31/PECAM-1 in the bEND.3 mouse endothelioma cell line by Western blot.

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Advantages of Using Secondary Antibodies

Bio-Techne primary and secondary antibodies were used to detect ACE-2 and CD14 in human PBMCs by flow cytometry.
Detection of ACE-2 and CD14 in human PBMCs by flow cytometry.

Indirect detection of target analytes using the combination of primary and secondary antibodies offers multiple advantages over directly detecting the proteins using only conjugated primary antibodies.

•  Amplifies the Signal

Multiple secondary antibodies can bind to a single primary antibody. Each bound secondary antibody is conjugated to a reporter molecule. This results in an amplification of the signal and an increase in the sensitivity of the assay. For some assays, the signal can be increased further using a biotinylated secondary antibody and the streptavidin-conjugated reporter molecule.

• Allows for Multiplexing Experiments

Since secondary antibodies are species specific, experiments can be designed to detect multiple analytes using primary antibodies derived from different species. The different analytes are detected using secondary antibodies conjugated to fluorochromes that emit light at different wavelengths.

• Preserves the Paratope

Conjugating a primary antibody to a reporter molecule has the potential of compromising its antigen-binding site, also called its paratope. This would reduce the primary antibody’s ability to bind to its target antigen. Using a conjugated secondary antibody allows an experimenter to use an unconjugated primary antibody in their assay.

Associated Products

Bio-Techne offers R&D Systems secondary antibodies conjugated to the VisUCyte HRP Polymer for cleaner, faster IHC results.

VisUCyte™ HRP Polymer

Bio-Techne’s VisUCyte HRP Polymer Antibodies eliminate the need to use Avidin-Biotin detection chemistry, thus providing cleaner and faster IHC staining of your tissue.

Bio-Techne offers R&D Systems Cell & Tissue Staining Kits were used to get optimized staining of GALNT10 in mouse testis tissue.

Cell & Tissue Staining Kits

Bio-Techne’s Cell & Tissue Staining Kits are ready-to-use kits that contain all the reagents needed for IHC staining of tissue sections and cytospin preparations.

Bio-Techne offers a wide variety of isotype control options that cover multiple species, isoforms and conjugates to match primary antibodies.

Isotype Control Antibodies

Isotype control antibodies are negative controls that help researchers interpret their results. Bio-Techne offers a wide range of isotype control antibodies to match the species, isoform, and conjugation of your primary antibody.

Additional Resources

Novus Biologicals Secondary Antibody Handbook provides valuable information to help you choose a secondary antibody for your experiments.

Secondary Antibodies Handbook

This handbook offers information you need for choosing the best secondary antibody for your experiment.

View of the Flow cytometry handbook and a single cell as a background

Flow Cytometry Handbook

This easy-to-follow guide provides an overview of the flow cytometry technique, as well as step-by-step protocols and troubleshooting tips.

View of the Immunohistochemistry (IHC) Handbook from BioTechne

Immunohistochemistry (IHC) Handbook

This comprehensive guide provides an introduction to the techniques, protocols, and troubleshooting methods that lead to a successful IHC experiment.