How does ECL HRP work?
ECL Uses an HRP Substrate for High-Sensitivity Western Blot Detection. Chemiluminescence assays are commonly used for the detection of proteins on western blots. The HRP substrate luminol emits light at 428 nm; however, luminol emits light only weakly, so enhancers are added to the reaction to increase the signal.
What does HRP react with?
Horseradish peroxidase (HRP) catalyzes the oxidation of substrates by hydrogen peroxide, resulting in a colored or fluorescent product and the release of light as a by-product of the reaction. HRP functions optimally at a near-neutral pH and can be inhibited by cyanides, sulfides and azides.
What does HRP do in Western blot?
HRP is often used in conjugates (molecules that have been joined genetically or chemically) to determine the presence of a molecular target. For example, an antibody conjugated to HRP may be used to detect a small amount of a specific protein in a western blot.
What does HRP bind to?
HRP is typically conjugated to an antibody, protein A, protein G, or avidin, although HRP can readily be conjugated to a wide range of different types of molecules.
What color is HRP?
Colorimetric substrates for ELISA applications
| Substrate | Enzyme | Absorbance and color |
|---|---|---|
| OPD Substrate | HRP | 490 nm (450 nm) Green (Orange) |
| PNPP (p-Nitrophenyl Phosphate) | ||
| 1-Step PNPP Substrate Solution | AP | 405 nm Yellow |
| PNPP Substrate | AP | 405 nm Yellow |
What does HRP do in ELISA?
One commonly used enzyme conjugate in ELISA is horseradish peroxidase. Horseradish peroxidase (HRP) is a 40,000 Dalton protein, which catalyzes the reduction of hydrogen peroxide (H2O2) to water (H2O) [1].
Is DAB a chromogen?
DAB (3,3′-Diaminobenzidine) is a derivative of benzene. It is most often used in immunohistochemical (IHC) staining as a chromogen. In DAB staining, DAB is oxidized by hydrogen peroxide in a reaction typically catalyzed by horseradish peroxidase (HRP).
What kind of light is produced by HRP?
HRP catalyzes the conversion of chromogenic substrates (e.g., TMB, DAB, ABTS) into colored products, and produces light when acting on chemiluminescent substrates (e.g. Enhanced Chemiluminescence by luminol ). Some of the most common HRP chromogenic substrates.
How can HRP catalyzed luminol CL intensity be increased?
As mentioned above, the HRP catalyzed luminol CL intensity can be greatly increased by adding certain compounds known as enhancers.
What kind of substrates does HRP work on?
These substrates fall into several distinct categories. HRP catalyzes the conversion of chromogenic substrates (e.g., TMB, DAB, ABTS) into colored products, and produces light when acting on chemiluminescent substrates (e.g. Enhanced Chemiluminescence by luminol). Some of the most common HRP chromogenic substrates.
What happens to luminol in horseradish peroxidase?
Horseradish peroxidase catalyses the oxidation of luminol to 3-aminophthalate via several intermediates. The reaction is accompanied by emission of low-intensity light at 428 nm. The reaction is accompanied by emission of low-intensity light at 428 nm.
