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Secondary antibodies are raised against the isotype of the primary antibody and are used to detect the primary antibody by binding to it in more than one place. This results in several secondary antibodies being bound to the primary antibody, each with multiple labels such as Alexa Fluor®, HRP, AP, Biotin, and gold conjugated secondary antibodies.
Many factors may affect the specificity, sensitivity, and consistency of your secondary antibodies. Here are some things to consider:
The secondary antibody is raised against the host species used to generate the primary antibody, for instance, if you use a primary antibody raised in rabbit, you will need an anti-rabbit secondary antibody raised in a host species other than rabbit (e.g. donkey anti-rabbit secondary).
Secondary antibodies tend to come in conjugated forms and. can be conjugated to
to enzymes, biotin, and fluorescent dyes/proteins.
The conjugate choice depends upon the experimental application and allows colorimetric, fluorescent, or chemiluminescent detection of primary antibodies in cell imaging, immunohistochemistry, flow cytometry, and western blotting.
For applications such as ELISA or western blotting, enzyme linked secondaries tend to be the most popular, whereas for flow cytometry or immunofluorescence, there is a preference for secondary antibodies conjugated to fluorescent proteins or dyes such as Alexa Fluor®.
The table below shows the most commonly used applications for different conjugated secondary antibodies:
Secondary antibodies must be directed against the isotype of the primary antibody. Polyclonal primaries are generally raised in rabbit, goat, sheep, or donkey and are generally IgG isotypes. The secondary antibody will therefore typically be an anti-IgG H&L (Heavy & Light chains) antibody.
Monoclonal primary antibodies are commonly raised in mouse, rabbit and rat; if the primary monoclonal antibody is a mouse IgG1, you will need an anti-mouse IgG or a less specific F(ab) fragment anti-mouse IgG.
Polyclonals bind several epitopes which results in greater sensitivity, although this can sometimes mean less specificity. Monoclonals detect a single epitope and result in highly specific antibodies.
We also offer recombinant multiclonal antibodies, which are a robust replacement for polyclonals. They’re a defined mixture of carefully selected individual recombinant monoclonal antibodies designed to recognize different epitopes on the same antigen, providing the same specificity and reproducibility as recombinant monoclonal antibodies.
Pre-adsorbed secondary antibodies
Pre-adsorbed secondary antibodies are ideal for multi-color experiments when several primary antibodies and their corresponding secondary antibodies are used simultaneously. The pre-adsorption process reduces the risk of cross reactivity between the secondary antibody and endogenous immunoglobulins present on cell and tissue samples.
F(ab) or (Fab')2 fragment secondary antibodies
F(ab) and (Fab')2 fragment antibodies eliminate non-specific binding between Fc portions of antibodies and Fc receptors on cells (such as macrophages, dendritic cells, neutrophils, NK cells and B cells), and penetrate tissues more efficiently due to their smaller size.
As fragment antibodies do not have Fc portions, they do not interfere with anti-Fc mediated antibody detection.
Biotinylated secondary antibodies
Biotin forms large complexes with avidin or streptavidin for signal amplification. Using either the avidin-biotin complex (ABC) method or labeled streptavidin-biotin (LSAB) method with biotinylated secondary antibodies can amplify signal in immunohistochemisty (IHC) or ELISA. Due to the conjugation of multiple biotin molecules to a secondary antibody, biotinylated secondary antibodies allow easy detection of proteins expressed at low levels. Learn more about Biotinylated secondary antibodies.
For more information on how to choose the right secondary antibody for your experiment, take a look at our secondary antibody selection guide.
Fluorescent labeled secondary antibodies
Fluorescent conjugates are preferred for cell imaging, flow cytometry, and immunohistochemistry frozen samples (IHC-Fr). They allow signal amplification detection of primary antibodies in cells and tissues due to an increased number of conjugated secondary antibodies that are able to bind to the primary antibody and are able to be detected by microscopy.
Enzyme labels are visualized with chromogenic reactions whereby a soluble colorless substrate is converted to a water-insoluble colored compound. Commonly used enzymes include horseradish peroxidase (HRP), used mostly in IHC, and alkaline phosphatase (AP), used mostly in western blot.
Absorption/emission spectrum and extinction coefficient
See below fluorescent conjugates absorption/emission spectrum and extinction coefficient to plan your multicolor panels, or click here to see our fluorochrome chart to help you to select the most appropriate fluorochrome:
Absorption Max (nm)
Emission Max (nm)
For more information on how to choose the right secondary antibody for your experiment, take a look at our secondary antibody selection guide: