Janelia Fluor® 549 Conjugated Antibodies
Janelia Fluor® 646 Conjugated Antibodies
HRP Conjugated Antibodies
Secondary Antibody Handbook
Secondary Antibody FAQs
Direct vs. Indirect Detection
Janelia Fluor® Dyes
What is a Secondary Antibody?
A secondary antibody is an antibody directed against another immunoglobulin (antibody) molecule. Under ideal conditions, a secondary antibody specifically binds the species and immunoglobulin (Ig) class (isotype) of the primary antibody in an immunoassay (i.e. indirect detection). The label, or conjugate, of the secondary antibody determines the method of detection, either chromogenic or fluorescent. In addition to detection of primary antibodies, secondaries are also used for antibody capture in ELISAs and for detection of recombinant protein.
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Secondary Antibody Production
Secondary antibody production involves harvesting from an animal that has been immunized with an antibody from another species. The specifications of the immunizing antibody (e.g. species, subclass, fragment, etc.) determine the specificity of the secondary antibody produced. For example, a goat anti-rabbit IgG (H+L) secondary antibody is produced by immunizing a goat with the whole IgG molecule or heavy and light chains of a rabbit IgG immunoglobulin molecule.
Why Use a Secondary Antibody?
Using a secondary antibody increases assay sensitivity and signal amplification due to the ability of multiple secondary antibodies to bind to a single primary antibody. The use of a secondary antibody also provides more flexibility in multiplexing (i.e. multiple labeling) experiments because commercially available secondary antibodies can be pre-conjugated to a wide range of labels.
Antibody Selection: Finding the Right Secondary
Factors to consider in secondary antibody selection:
- Host Species – the species used to raise the secondary antibody
- Format – the structure and fragment of the secondary antibody
- Species Reactivity – the species recognized by the secondary antibody
- Target Immunoglobulin – The primary antibody class/isotype or subclass
- Specificity – The region of the target immunoglobulin recognized by the secondary antibody
- Conjugate – the detection label attached to the secondary antibody
If you cannot find the product you’re looking for Novus Biologicals can help you find the right antibody in addition to providing custom services, such as custom conjugation, to meet your experimental needs.
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Example of Secondary Antibody Nomenclature
(Host of the Primary
(Class and subclass of
the Primary Antibody)
Heavy + Light chain,
Target Specificity (Region
of the Primary Antibody)
Step to Reduce Species
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Why Choose a Pre-adsorbed Secondary Antibody?
Pre-adsorption is a technique used to increase secondary antibody specificity and reduce non-specific binding. The pre-adsorbtion procedure works by passing antibodies through a column containing immobilized serum proteins or immunoglobulin from potentially cross-reactive species. The reactive antibodies bind to the proteins and immunoglobulins, while the non-reactive antibodies flow through the column.
Pre-adsorbed secondary antibodies are recommended when determining protein expression in multiplexing experiments or when staining immunoglobulin-rich tissues or cells in order to help prevent non-specific binding. It is advised to use a secondary antibody pre-adsorbed against the species of the experimental sample. For example, a secondary antibody adsorbed against rat immunoglobulin or serum is recommended when staining rat tissue.
Janelia Fluor® Conjugated Secondary Antibodies
Janelia Fluor® 549 and Janelia Fluor® 646 conjugated secondary antibodies expand the palette of bright, photostable dyes available for your super resolution microscopy techniques including dSTORM and STED imaging experiments.
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