Is horseradish peroxidase fluorescence?

Horseradish peroxidase uses Amplex red as an electron donor during the reduction of hydrogen peroxide to water. The resultant product, resorufin, is a highly colored and fluorescent compound. One commonly used enzyme conjugate in ELISA is horseradish peroxidase.

What is the purpose of horseradish peroxidase?

Horseradish peroxidase (HRP) is an enzyme used to amplify signal in photometric assays by catalyzing the conversion of chromogenic or chemiluminescent substrates for the detection of targets such as proteins, carbohydrates, and nucleic acids.

How does horseradish peroxidase HRP contribute to creating the final colored sample?

Question: How does horseradish peroxidase (HRP) contribute to creating the final colored sample? A. HRP is a protein that becomes colored when bound to an antibody associated with its epitope. HRP is an enzyme that catalyzes the conversion of a substrate to a colored product.

What is a horseradish peroxidase assay?

Introduction. Horseradish peroxidase (HRP) is an enzyme that when in the presence of hydrogen peroxide, oxidizes a substrate (oxygen donor), resulting in a color change that is quantifiable. There are about 40 HRP isoenzymes.

What color is horseradish peroxidase?

Tetramethylbenzidine (TMB) and similar substrates are oxidized by HRP in the presence of hydrogen peroxide, or nontoxic substitutes, to yield a chinoid structure of intense blue color.

What is the substrate for peroxidase?

Optimal substrates For many of these enzymes the optimal substrate is hydrogen peroxide, but others are more active with organic hydroperoxides such as lipid peroxides. Peroxidases can contain a heme cofactor in their active sites, or alternately redox-active cysteine or selenocysteine residues.

What is the biological role of HRP in horseradish?

The enzyme horseradish peroxidase (HRP), found in the roots of horseradish, is used extensively in biochemistry applications. It is a metalloenzyme with many isoforms, of which the most studied type is C. It catalyzes the oxidation of various organic substrates by hydrogen peroxide.

Is HRP an oxidizing agent?

3.1 Direct electron transfer of HRP. HRP can react with H2O2 to form a powerful enzymatic oxidizing agent known as Compound I, which is a two-equivalent oxidized form containing an oxyferryl heme (Fe4+=O) and a porphyrin φ cation radical.

How does TMB work?

Background: TMB (3,3′, 5,5”-tetramethylbenzidine) is used as a substrate to generate detectable signal in ELISA. The reaction between the TMB substrate and a peroxidase, typically horseradish peroxidase (HRP), produces a measur- able color change that correlates with analyte level.

How is the horseradish peroxidase enzyme conjugate detected?

Alone, the HRP enzyme, or conjugates thereof, is of little value; its presence must be made visible using a substrate that, when oxidized by HRP using hydrogen peroxide as the oxidizing agent, yields a characteristic color change that is detectable by spectrophotometric methods.