Detection Using Horseradish Peroxidase HRP

Horseradish peroxidase catalyzes the cleavage of hydroperoxide substrates forming active oxygen, which oxidizes molecules resulting in a colored product. For application in Western blots the reaction product must be insoluble in aqueous buffer solutions. 

50 mg/ml4-chloro-l-naphthol (4-CN), 3,3, 4,4 -tetraaminodi-phenyl ether, or 3,3 -diaminobenzidine (DAB) in DMF (stock solution) 

B 600 pi 0.1% H202-urea adduct (w/v) in ddH20, 10 pi 10% CUSO4 (w/v), 5 pi 10% NiS04 (w/v) or NiCl2 and 9.35 ml PBS are mixed, then 40 pi of Soln. A are added. Prepare reagent immediately before use. 

E 20 mM ammonium molybdate ((NH4)6Mo7024 4H2O, MW 1235.86) in ddH20. Stable at RT. 

1% H202-urea adduct (10.6 mM H2O2) in ddH20. Stable for several days in a refrigerator. 

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