Horse Radish Peroxidase (HRP) Mechanism of Action

Horse Radish Peroxidase(HRP) is heme-containing protein isolated from wild horse radish roots.HRP catalyzes the transfer of two electrons from a substrate to hydrogen peroxide(H₂O₂), to generate H₂O and an oxidized donor. When 3',3'-diaminobenzidine(DAB) is used as a substrate, the oxidized product polymerizes to form a brown precipitate in water and ethanol. In presence of cobalt or nickel the product will be blue-black. This form the basis of chromogenic assays for peroxidase in immunochemistry, western blotting etc.

Horse Radish Peroxidase Mechanism - Developing a western blot / Elisa

Developing a western blot:

Developing an immunoblot of HRP conjugated antibody is explained here, developing solution is made by dissolving the substrate 0.03% diaminobenzidene(DAB) in PBS and 1% CoCl_2,   to this 0.1% hydrogen peroxide (H₂O₂)is added prior to the incubation with the membrane. even preformulated DAB solutions are readily available. Wash the membrane with wash buffer and incubate with developing solution for 30 seconds to one minute,DAB reacts with HRP in the presence of peroxide to yield an insoluble brown-colored product at locations where peroxidase-conjugated antibodies are bound to the target protein. The reaction can be stopped by adding water.

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Reference:

Molecular Cloning, J.Sambrook