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.

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

Normal Phase & Reverse Phase Chromatography

Normal Phase
In a very non-polar environment, hydrophilic molecules will tend to associate with each other (like water drops on an oily surface). The hydrophilic molecules in the mobile phase will tend to adsorb to the surface on the inside and outside of a particle if that surface is also hydrophilic. Increasing the polarity of the mobile phase will subsequently decrease the adsorption and ultimately cause the sample molecules to exit the column. This mechanism is called Normal Phase Chromatography. It is a very powerful technique that often requires non-polar solvents. Due to safety and environmental concerns this mode is used mostly as an analytical technique and not for process applications.

Reversed Phase
The opposite of normal phase, or Reversed Phase Chromatography, results from the adsorption of hydrophobic molecules onto a hydrophobic solid support in a polar mobile phase. Decreasing the mobile phase polarity by adding more organic solvent reduces the hydrophobic interaction between the solute and the solid support resulting in de-sorption. The more hydrophobic the molecule the more time it will spend on the solid support and the higher the concentration of organic solvent that is required to promote de-sorption.

Popularity
Reversed phase chromatography (RPC) is the most popular separation technique at analytical scale, because:
RPC applies to a very wide range of molecules including charged and polar molecules,
RPC allows precise control of variables such as organic solvent type and concentration, pH, and temperature,
RPC columns are efficient and stable,
RPC is a robust technique.

At process scale, RPC is not typically used for protein purification due to the presence of the organic solvent which can cause denaturation of proteins and destroys their biological activity. Reversed phase chromatography is the most dominant analytical HPLC technique and there are many different stationary phases available for method optimization.

Source:
Tosoh Bioscience Resource Centre