Specialising in Secondary Antibodies and Conjugates
What is a secondary antibody?
A secondary antibody is one that recognizes an antibody or antibody domain from a different species. Secondary antibodies are used to bind primary antibodies (specific for a protein of interest (antigen)) in many different experimental schemes. Conjugated secondary antibodies (labeled with reporter molecules such as enzymes or fluorophores) can be used to visualize the antigen. There are many applications for unconjugated secondary antibodies as well, for example, coating ELISA plates or beads for capturing and/or purifying primary antibodies.
Fluorescent probes or fluorophores (fluorescent dyes or proteins) are coupled to a secondary antibody or streptavidin to allow visualization of an analyte. Each fluorophore has its specific spectral characteristics, with excitation and emission spectra particular to the molecule. Use the spectra viewer to build dye panels and compare the suitability of dyes for your application.
Immunoglobulins from different species share similar structures. Secondary antibodies raised against one species are likely to recognize epitopes on other species’ immunoglobulins through those shared structures. This cross-reactivity can cause background tissue staining or signal from a non-target primary antibody.
Like colorimetric and chemiluminescent Western blotting, fluorescent Western blotting uses the antigen-antibody complex to detect specific proteins immobilized on a blotting membrane after separation by electrophoresis. Visualization of the protein is achieved by exciting the fluorescent dye using an imaging system equipped with an appropriate light source.
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Chemiluminescent Western blotting
