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Horseradish peroxidase is a 44,173.9-dalton glycoprotein with six lysine residues which can be conjugated to a labeled molecule. It produces a coloured, fluorimetric [ 6 ] or luminescent derivative of the labeled molecule when incubated with a proper substrate, allowing it to be detected and quantified.
Secondary antibodies can be conjugated to enzymes such as horseradish peroxidase (HRP) or alkaline phosphatase (AP); or fluorescent dyes such as fluorescein isothiocyanate (FITC), rhodamine derivatives, Alexa Fluor dyes; or other molecules to be used in various applications.
In the case of electron microscopy, antibodies are linked to a heavy metal particle (typically gold nanoparticles in the range 5-15nm diameter). As previously described, enzymes such as horseradish peroxidase or alkaline phosphatase are commonly used to catalyse reactions that give a coloured or chemiluminescent product.
This means that several secondary antibodies will bind to one primary antibody and enhance the signal, allowing the detection of proteins of a much lower concentration than would be visible by SDS-PAGE alone. Horseradish peroxidase is commonly linked to secondary antibodies to allow the detection of the target protein by chemiluminescence.
This primary antibody could be in the serum of a donor, to be tested for reactivity towards the antigen. Enzyme-linked secondary antibodies are applied as detection antibodies, which bind specifically to the antibody's Fc region (nonspecific). The plate is washed to remove the unbound antibody-enzyme conjugates.
Immunocytochemistry labels individual proteins within cells, such as TH (green) in the axons of sympathetic autonomic neurons.. Immunocytochemistry (ICC) is a common laboratory technique that is used to anatomically visualize the localization of a specific protein or antigen in cells by use of a specific primary antibody that binds to it.