- Horseradish peroxidase (HRP) is widely used in immunoassays and histological techniques because of its ability to catalyze the oxidation of various chromogenic, fluorogenic, and chemiluminescent substrates in the presence of hydrogen peroxide.
- The choice of substrate determines the type of signal produced—visible color, fluorescence, or light emission—and thus influences the sensitivity, stability, and application of the assay.
- Common substrates such as DAB, TMB, AEC, and luminol are selected based on the specific requirements of methods like immunohistochemistry, ELISA, or Western blotting.
- Understanding their properties allows researchers to match detection systems with experimental goals for optimal results.
- Here’s a list of the most common HRP substrates, with their signal type, color, and typical applications:
3,3′-Diaminobenzidine (DAB)
- Signal/Color: Brown, insoluble precipitate.
- Application: Widely used in immunohistochemistry (IHC) and immunocytochemistry (ICC). The brown stain is permanent and stable, making it suitable for light microscopy and long-term slide storage.
3-Amino-9-ethylcarbazole (AEC)
- Signal/Color: Red, insoluble precipitate.
- Application: Alternative to DAB in IHC/ICC when contrast against tissue background is preferred. However, AEC is alcohol-soluble, so aqueous mounting media are required.
Tetramethylbenzidine (TMB)
- Signal/Color: Blue (soluble in aqueous solution; can turn yellow upon acid stop reaction).
- Application: Standard substrate in ELISA assays due to its strong, quantifiable color change. The reaction is usually stopped with sulfuric or phosphoric acid, stabilizing the yellow product for spectrophotometric measurement.
o-Phenylenediamine dihydrochloride (OPD)
- Signal/Color: Orange/yellow soluble product.
- Application: Formerly common in ELISA, but less used today due to safety concerns and the superior performance of TMB.
Luminol (and Enhanced Chemiluminescence substrates, ECL)
- Signal/Color: Light emission (blue/white glow).
- Application: Western blotting and highly sensitive assays. HRP catalyzes luminol oxidation, producing chemiluminescence captured by X-ray film or digital imaging systems. Enhanced substrates (e.g., with p-iodophenol) improve intensity and duration of signal.
4-Chloro-1-naphthol (4-CN)
- Signal/Color: Blue-purple insoluble precipitate.
- Application: Used for membrane-based assays such as dot blots and Western blots, though less stable than DAB.
Tyramide Signal Amplification (TSA) substrates
- Signal/Color: Fluorescent or chromogenic, depending on tyramide conjugate.
- Application: IHC, ICC, and in situ hybridization where very low-abundance targets need detection. HRP catalyzes the deposition of labeled tyramides directly at the site of the antigen, amplifying signal intensity dramatically.
