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. 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. HRP conjugates are used as reporters in a number of common techniques including western blotting, enzyme-linked immunosorbent assays (ELISAs), and microscopy.
HRP conjugates are commonly used in indirect assays. The horseradish peroxidase enzyme conjugate does not bind directly to the target but rather to an antibody or other molecule that has been bound to the target in a previous step. The indirect method has two advantages: the two-step procedure provides greater amplification because there are usually multiple binding sites for the conjugate on the molecule previously bound to the target, and the conjugate does not need to be specific for the target, but only for the molecule type bound to the target, saving time and money. For instance, an anti-mouse-HRP conjugate can be used in any assay where the primary antibody is mouse, or a protein A-HRP conjugate can be used for any biotinylated primary antibody.
HRP Substrate Types
There are several different types of HRP substrates. The type of substrate used for detection depends on the particular assay and mode of detection. Chromogenic HRP substrates, which remain in solution and become colored after reaction with HRP, are often used for ELISAs and other colorimetric assays. Commonly used chromogenic HRP substrates include 3,3',5,5'-tetramethylbenzidine (TMB) and 2,2' -azino-di-[3-ethylbenzthiazoline-6-sulfonic acid] (ABTS). The choice of soluble chromogenic substrate depends on the requirements for assay sensitivity and the capabilities of the microplate reader.
After oxidation by HRP, some substrates will precipitate. An oxidized HRP substrate can form a precipitate that is colored, electron-dense, or luminescent. An example of an HRP substrate that is both is colored and electron-dense is 3,3'-diaminobenzidine (DAB), which can be used in western blotting and both light and electron microscopy. In a chemiluminescence assay, widely used for western blotting and less frequently for immunocytochemistry, a luminescent HRP substrate emits light after oxidation by HRP.
After oxidation by HRP, some substrates will precipitate. Substrates that form a precipitate can be colored, electron-dense, or luminescent. An example of a substrate that is both is colored and electron-dense is 3,3'-diaminobenzidine (DAB), which can be used in western blotting and both light and electron microscopy. In a chemiluminescence assay, widely used for western blotting and less frequently for immunocytochemistry, a luminescent substrate emits light after oxidation by HRP.
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. This enhancement of a luminol-based signal is commonly referred to as enhanced chemiluminescence (ECL).
There are a number of different enhancers available, some of which can increase the signal by as much as a 1,000-fold, making ECL more sensitive than other common detection systems such as conversion of substrates to colored precipitates. The type of enhancer used can also increase the signal duration. Luminol has an emission half-life of less than a minute. The Bio-Rad ECL system extends the signal duration to 24 hours, permitting reimaging without loss of signal intensity.
The advantages of using Bio-Rad HRP substrate-based ECL include:
- Sensitive detection at the femtogram level
- Low background signal
- Long signal duration
- Compatible with any digital or film-based imaging protocol and instrument
- Can be used with any HRP conjugate
- Long shelf life at room temperature