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Horseradish Peroxidase (HRP)

The enzyme horseradish peroxidase (HRP), found in the roots of horseradish, is used extensively in biochemistry applications primarily for its ability to amplify a weak signal and increase detectability of a target molecule. HRP is often used in conjugates (molecules that have been joined genetically or chemically) to determine the presence of a molecular target. HRP is also commonly used in techniques such as ELISA and immunohistochemistry due to its monomeric nature and the ease with which it produces colored products. Peroxidase, a heme-containing oxidoreductase, is a commercially important enzyme which catalyzes the reductive cleavage of hydrogen peroxide by an electron donor.

Horseradish peroxidase is ideal in many respects for these applications because it is smaller, more stable, and less expensive than other popular alternatives such as alkaline phosphatase. It also has a high turnover rate that allows generation of strong signals in a relatively short time span. It should be noted that high concentrations of phosphate severely decrease stability of horseradish peroxidase. In addition to biomedical applications, horseradish peroxidase is one of the enzymes with important environmental applications. This enzyme is suitable for the removal of hydroxylated aromatic compounds (HACs) that are considered to be primary pollutants in a wide variety of industrial wastewater.

 

ReadiUse™ Colorimetric HRP Substrate Solutions


HRP and HRP conjugates facilitate the ABTS oxidation in the presence of hydrogen peroxide, turning ABTS into its blue-green oxidized product. This chromogenic reaction is widely used to quantify HRP in ELISA assays. The oxidized ABTS product has the absorption maximum at 420 nm that can easily be followed with a spectrophotometer. ReadiUse™ ABTS Substrate Solution (Cat# 11001) is optimized for ELISA assays that use HRP or HRP-labeled conjugates and hydrogen peroxide in microwell plates or test tubes. Our ABTS solution allows HRP reaction to be done with a single addition. The assay solution changes its color to light green upon its reaction with HRP or HRP conjugates.

ReadiUse™ TMB Substrate Solution (Cat# 11003) is a premixed solution of TMB substrate with hydrogen peroxide. It produces a blue product upon interaction with HRP or HRP conjugates without the addition of hydrogen peroxide. The soluble blue product can be quantitated at 650 nm. The use of a stop solution produces 2-4 fold increase in sensitivity and the resulting yellow solution can be read at 450 nm. ReadiUse™ TMB Substrate Solution provides a convenient and ultrasensitive quantitative substrate system.


Left: HRP dose responses were measured with ReadiUse™ ABTS Substrate Solution (Cat#11001) in a 96-well clear plate using a SpectraMax® microplate reader (Molecular Devices). Right: HRP dose responses were measured with ReadiUse™ TMB Substrate Solution (Cat# 11003) in a 96-well clear plate using a SpectraMax® microplate reader (Molecular Devices). As low as 3 µU/well peroxidase was detected with 10 minutes incubation.

 

ReadiUse™ Hydrogen Peroxide Solution



HRP dose responses on a 384-well plate were measured with Amplite® ADHP (Cat# 11000) and ReadiUse™ Hydrogen Peroxide Solution (Cat# 11004).
Compared with other commercial hydrogen peroxide solutions, our formulated ReadiUse™ Hydrogen Peroxide Solution (Cat# 11004) is much more stable. It is calibrated to ensure more reproducible peroxidase-based assays.
 

Fluorimetric HRP Detection Substrates


A variety of substrates are available for detecting peroxidase activity in ELISA assays. Colorimetric HRP substrates (e.g., TMB, OPD and ABTS) have been widely used for years. Each of these substrates varies greatly with its performance characteristics such as detection sensitivity, working range and attainable signal-to-noise ratio. Amplite® Blue (Cat# 11005) is a soluble fluorogenic HRP substrate for the detection of peroxidase activity. Amplite® Blue allows rapid HRP detection assays to be performed with greater sensitivity than the colorimetric HRP substrates. The fluorescent product of Amplite® Blue does not photobleach. Amplite® Blue exhibits a flat baseline in assays, which facilitates low-level detection sensitivity and allows for high signal-to-noise ratio.

Amplite® IR (Cat# 11009) is a fluorogenic peroxidase substrate that generates near infrared fluorescence upon reaction with peroxidase and H2O2. It can be used to detect both H2O2 and peroxidase. Amplite® IR generates a substance that has maximum absorption at 647 nm with maximum emission at 670 nm. This near infrared absorption and fluorescence minimize the assay background often caused by the autoabsorption and/or autofluorescence of biological samples that rarely absorb light beyond 600 nm. Unlike other HRP substrates, such as dihydrofluoresceins and dihydrorhodamines, the air-oxidation of Amplite® IR is minimal. Compared with ADHP (also called Amplex® Red in literature), Amplite® IR generates the fluorescence that is pH-independent from pH 4 to 10. Thus it is a superior alternative to ADHP (Amplex® Red) for detections that require low pH where ADHP (Amplex® Red) has reduced fluorescence. We have used Amplite® IR to detect HRP in quite a few immunoassays. Amplite® IR can also be used to detect a trace amount of H2O2. Because H2O2 is produced in many enzymatic redox reactions, Amplite® IR can be used in coupled enzymatic reactions to detect the activity of many oxidases and/or related enzymes/substrates or cofactors such as glucose, acetylcholine, cholesterol, L-glutamate, and amino acids, etc.

Amplite® ADHP (Cat# 11000) is chemically similar to Amplex® Red (Amplex® Red is the trademark of Invitrogen). It is a sensitive fluorogenic peroxidase substrate. Amplite® ADHP has the highest quality of ADHP with much lower background than the materials from other commercial vendors. Amplite® ADHP generates highly fluorescent resorufin that has maximum absorption at 571 nm and maximum emission at 585 nm. Unlike other HRP substrates, such as dihydrofluoresceins and dihydrorhodamines, the air-oxidation of Amplite® ADHP is minimal. So far ADHP has been known as the most sensitive and stable fluorogenic probe for detecting HRP and H2O2. ADHP has been widely used to detect HRP in many immunoassays. On the other hand, ADHP can also be used to detect a trace amount of H2O2. The ADHP-based H2O2 detection is at least one Because H2O2 is produced in many enzymatic redox reactions, ADHP can be used in coupled enzymatic reactions to detect the activity of many oxidases and/or related enzymes/substrates or cofactors such as glucose, acetylcholine, cholesterol, L-glutamate and amino acids, etc.


Left: H2O2 dose responses measured in a solid black 96-well plate with Amplite® Blue (Cat# 11005). Center: H2O2 dose responses measured in a solid black 96-well plate with Amplite® Fluorimetric Hydrogen Peroxide Assay Kit (Cat# 11502). As low as 0.03 µM H2O2 was detected. Right: H2O2 dose responses measured in a solid blakc 384-well plate with Amplite® Fluorimetric Hydrogen Peroxide Assay Kit (Cat# 11501). As low as 0.03 µM H2O2 was detected.

 

HRP Reaction Stopping Solution



Application of Signal Guard™ HRP Reaction Stopping Solution (Cat# 11020) on HRP coupled glucose detection reaction. Reactions were incubated at room temperature for 5 minutes and then 20 µL 1X stop reagent was added, and the reaction was completely inhibited by Signal Guard™ HRP Reaction Stopping Solution.
HRP coupling reactions provide sensitive biomolecular assays based on hydrogen peroxide"generating enzyme systems linked to peroxidase"mediated oxidation. Fluorogenic HRP substrates are preferred for enhancing assay sensitivity. Among them, the most commonly used HRP substrates include ADHP (also called Amplex® Red), Amplex® UltraRed and Amplite® Red. Typically, detection reactions are performed in microplate wells and are initiated by adding a fluorogenic HRP substrate, resulting in continuous fluorescence increase. It is critical to ensure that the timing of the standard and unknown sample measurements is the same. Signal Guard™ HRP Reaction Stopping Solution (Cat# 11020) provides convenience and control by allowing the fluorescence signal-generating reaction to be terminated at a user-determined time point. After the addition of the stop reagent, the fluorescence signal remains stable. The Signal Guard™ HRP Reaction Stopping Solution is designed to be used in conjunction with ADHP (Amplex® Red ), Amplite® Red and Amplex® UltraRed fluorogenic substrates. It can also be used in other HRP reaction systems.

 

HRP Conjugation Tools


ReadiUse™ Preactivated HRP NHS Ester (Cat# 11025) is the mono NHS ester of HRP. It can be used to readily label proteins (such as antibodies) or other biological molecules that have an amino group. ReadiUse™ Preactivated HRP NHS Ester is robust and easy to use. It can used to label antibodies with a simple mixing, i.e. the basic solution of an antibody (pH 8.5-9.0) can be directly mixed with the HRP NHS ester and shaken for 1-2 hours. In most cases, the resulting solution can be directly used for ELISA assays without further purification.


The labeling principles of HRP NHS ester (top) and HRP maleimide (bottom) with proteins and other biomolecules.
ReadiUse™ Preactivated HRP Maleimide (Cat# 11026) is a thiol-reactive HRP derivative. It can be used to readily label proteins (such as antibodies) or other biological molecules that have a thiol group. ReadiUse™ Preactivated HRP Maleimide is robust and easy to use. It can be used to label antibodies with a simple mixing, i.e. the slightly acidic or neutral solution of an antibody (pH 6-7) can be directly mixed with the HRP maleimide, and the reaction mixture is shaken for 1-2 hours. In most cases, the resulting solution can be directly used for ELISA assays without further purification.


The working principle of ReadiLink™ HRP Antibody Conjugation Kits (Cat# 5503 & 5504).


Protein-protein conjugations are commonly performed with a bifunctional linker (such as the commonly used SMCC), which has different reactivity on each end for linking two different proteins. One end of the crosslinker reacts (via NHS ester) with amines (-NH2) found in the amino acid lysine and N-terminus, and the other end reacts (via maleimide) with the thiol groups (-SH) found in the amino acid cysteine. SMCC-modified proteins are extremely unstable and often self-reactive since proteins often contain both amine and thiol groups that cause a significant amount of homo-crosslinking. In addition, it is quite difficult and tedious to quantify the number of maleimide groups on a protein. ReadiLink™ Peroxidase (HRP) Antibody Conjugation Kits (Cat# 5503 & 5504) are designed for preparing horseradish peroxidase (HRP) conjugates directly from proteins, peptides, and other ligands that contain a free amino group. The HRP provided in our kits has been pre-activated with our proprietary linker Buccutite™ FOL, and can be directly used for conjugation. The Buccutite™ FOL-activated HRP readily reacts with Buccutite™ MTA-containing molecules under mild neutral conditions without any catalyst required. Compared with commonly used SMCC and other similar technologies, our Buccutite™ bioconjugation system is much more robust and easier to use. It enables faster and quantitative conjugation of biomolecules with higher efficiencies and yields. Kit 5503 is optimized for labeling 100 µg protein, and Kit 5504 is optimized for 1 mg protein.


Goat Anti-Rabbit conjugate with Signal Guard™ HRP Conjugate Stabilizer (Cat# 11010) was stored at 4 °C and tested over 50 weeks using Amplite® ADHP (Cat# 11000).
The major advantage of a liquid HRP-conjugate is that it eliminates the chance for human error when dissolving or diluting the lyophilized or concentrated conjugate. Adding too much or not enough buffer results in assay to assay variation. Until now, the disadvantage of this format is the inherent instability of the liquid HRP-conjugates. Signal Guard™ HRP Conjugate Stabilizer (Cat# 11010) is formulated to allow you to provide pre-diluted, ready-to-use conjugates. This formulation can be used to create stock solutions (1:1,000) or ready-to-use assay reagents (1:100,000) that can be stored at 4°C for 24 months or at room temperature for six months.
 

Table 1. HRP (Horseradish Peroxidase) Detection Probes and Assays

Cat No.
Product Name
Ex (nm)
Em (nm)
Unit Size
Price
11000Amplite® ADHP [10-Acetyl-3,7-dihydroxyphenoxazine] *CAS#: 119171-73-2*57158525 mg$217
11005Amplite® Blue32440925 mg$162
11502Amplite® Fluorimetric Hydrogen Peroxide Assay Kit *Near Infrared Fluorescence*647670500 Tests$272
11009Amplite® IR6476701 mg$106
11011Amplite® Red5715851000 Assays$106
5503Buccutite™ Peroxidase (HRP) Antibody Conjugation Kit *Optimized for Labeling 100 ug Protein* 2 Labelings$259
5504Buccutite™ Peroxidase (HRP) Antibody Conjugation Kit *Optimized for Labeling 1 mg Protein* 1 Labeling$521
11001ReadiUse™ ABTS Substrate Solution *Optimized for ELISA Assays with HRP Conjugates*420 1 L$194
11004ReadiUse™ hydrogen peroxide solution *50 mM calibrated and stabilized solution* 5 X 10 mL $106
11026ReadiUse™ Preactivated HRP maleimide 100 µg $106