Secondary antibodies can be conjugated to a large number of labels, including enzymes, biotin, and fluorescent dyes/proteins. The label of choice depends upon the experimental application.
|Immunoassay experiment||Secondary antibody label|
|ELISA||Enzymes (usually HRP or AP), biotin|
|Immunofluorescence||Fluoresecent label, biotin|
|Flow cytometry||Fluorescent label, biotin|
|Immunocytochemistry||Fluorescent label, biotin|
Enzyme labels are visualized with chromogenic reactions whereby a soluble colorless substrate is converted to a water-insoluble colored compound.
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
Alkaline phosphatase (AP) is a hydroylase enzyme and its signal is often measured through its colorimetric substrate p-nitrophenyl phosphate (pNPP).
Multiple biotin molecules can be conjugated to a secondary antibody, providing an amplification step that makes this conjugate 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 multi-color analysis. A single dye is excited at a particular wavelength and emits a photon at another wavelength. Alternatively, a tandem dye consists of a donor and acceptor fluorochrome placed in close proximity, allowing energy transfer between the two. The tandem dye is excited at the excitation wavelength of the donor molecule and emits a photon at the emission wavelength of the acceptor molecule.
The table below lists fluorescent labeled secondary antibodies within our portfolio.
|Label||Absorption Max (nm)||Emission Max (nm)||Extinction Coefficient|
|Alexa Fluor® 405||402||421||35,000|
|Alexa Fluor® 488||495||519||73,000|
|Alexa Fluor® 555||555||565||155,000|
|Alexa Fluor® 568||578||603||88,000|
|Alexa Fluor® 594||590||617||92,000|
|Alexa Fluor® 647||650||668||270,000|
|Alexa Fluor® 680||679||702||183,000|
|Alexa Fluor® 750||749||775||290,000|
|Alexa Fluor® 790||784||814||260,000|
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.