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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.
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.
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.
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.
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.
The antigens are either from cell extracts or recombinant. Blood serum is incubated in the wells of the plate and is washed out. If antibodies that bind to antigen are present then they will remain after washing. A secondary anti-human antibody conjugated to an enzyme such as horseradish peroxidase is added. The enzyme reaction will produce a ...
The secondary antibody is tagged with peroxidase. Optimal staining depends on a number of factors including the antibody dilution, the staining chemicals, the preparation and/or fixation of the cells/tissue, and length of incubation with antibody/staining reagents.
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.