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Secondary antibodies can be conjugated 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.
|Immunoassay experiment||Secondary antibody label|
|Flow cytometry||Alexa Fluor®, Phycoerythrin, FITC|
|Western blot||Enzymes - HRP or AP|
|ELISA||Enzymes - HRP or biotin|
|Electron microscopy, LFA||Gold Nanoparticles|
|See all secondary antibodies|
Multiple biotin molecules can be conjugated to a secondary antibody, biotinylated secondary antibody provides an amplification that makes biotin-conjugated secondary antibodies suitable for detecting proteins expressed at low levels. Visualization is through interaction between biotin and streptavidin, where the streptavidin is bound to labels such as HRP or fluorescent probes. During the assay, it is normal procedure for the biotin-labeled secondary antibody to be added first, sequentially followed by the streptavidin conjugated to a label.
Fluorescent conjugates are preferred for cell imaging, flow cytometry, and immunohistochemistry frozen samples (IHC-Fr). A single dye is excited at a particular wavelength and emits a photon at another wavelength, detected by a fluorescent microscope. Fluorescent conjugate secondary antibodies allow signal amplification detection of primary antibodies in cells and tissues due to increase 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. Some enzymes, like HRP and AP, allow colorimetric or chemiluminescent detection of primary antibodies in applications like western blot and immunohistochemistry.
Commonly used enzymes:
Horseradish peroxidase (HRP) is visualized by chromogenic reactions such as diaminobenzidine (DAB) or chemiluminescence. HRP is a 44kDa glycoprotein enzyme label and is more stable than alkaline phosphatase.
HRP labeled secondary antibodies
HRP-polymer secondaries by binding more horseradish peroxidase than standard HRP secondary antibodies allows amplification of signal ideal for those low expressing targets in IHC.
Alkaline phosphatase (AP) is a hydrolase enzyme and its signal is often measured through its colorimetric substrate p-nitrophenyl phosphate (pNPP). Used mostly in western blot.
See below fluorescent conjugates absorption/emission spectrum and extinction coefficient to plan your multicolor panels.
Absorption Max (nm)
Emission Max (nm)
Alexa Fluor® and Texas Red® are registered trademarks of Life Technologies. Alexa Fluor® dye conjugates contain(s) technology licensed to Abcam by Life Technologies. DyLight® is a registered trademark of Thermo Fisher Scientific Inc and its subsidiaries.