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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 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.
TMB can act as a hydrogen donor for the reduction of hydrogen peroxide to water by peroxidase enzymes such as horseradish peroxidase.. Shows the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) to 3,3',5,5'-tetramethylbenzidine diimine
It is commonly used as a substrate with hydrogen peroxide for a peroxidase enzyme (such as horseradish peroxidase) or alone with blue multicopper oxidase enzymes (such as laccase or bilirubin oxidase). Its use allows the reaction kinetics of peroxidases themselves to be followed.
Peroxidase sequences come from other general public databases (NCBI, TIGR, UniProt KnowledgeBase: all the databases used are listed in the PeroxiBase), either as pre-existing annotated sequences, or from raw data (whole genomic sequencing projects).
Horseradish peroxidase has an accessible active site, and many compounds can reach the site of the reaction. On the other hand, for an enzyme such as cytochrome c peroxidase, the compounds that donate electrons are very specific, due to a very narrow active site.
Horseradish peroxidase is also capable of oxidizing these substrates, but its heme is not covalently bound and becomes damaged during turnover. [4] A specific vanadium bromoperoxidase in marine organisms (fungi, bacteria, microalgae, perhaps other eukaryotes) uses vanadate and hydrogen peroxide to brominate electrophilic organics. [5]
Streptavidin is widely used in Western blotting and immunoassays conjugated to some reporter molecule, such as horseradish peroxidase. Streptavidin has also been used in the developing field of Nanobiotechnology, the use of biological molecules such as proteins or lipids to create nanoscale devices/structures.