![]() ![]() This enhancement of a luminol-based signal is commonly referred to as enhanced chemiluminescence (ECL). 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. ECL Uses an HRP Substrate for High-Sensitivity Western Blot DetectionĬhemiluminescence assays are commonly used for the detection of proteins on western blots. In a chemiluminescence assay, widely used for western blotting and less frequently for immunocytochemistry, a luminescent substrate emits light after oxidation by HRP. 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. Substrates that form a precipitate can be colored, electron-dense, or luminescent. In a chemiluminescence assay, widely used for western blotting and less frequently for immunocytochemistry, a luminescent HRP substrate emits light after oxidation by HRP.Īfter oxidation by HRP, some substrates will precipitate. 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. An oxidized HRP substrate can form a precipitate that is colored, electron-dense, or luminescent. The choice of soluble chromogenic substrate depends on the requirements for assay sensitivity and the capabilities of the microplate reader.Īfter oxidation by HRP, some substrates will precipitate. Commonly used chromogenic HRP substrates include 3,3',5,5'-tetramethylbenzidine (TMB) and 2,2' -azino-di- (ABTS). ![]() Chromogenic HRP substrates, which remain in solution and become colored after reaction with HRP, are often used for ELISAs and other colorimetric assays. The type of substrate used for detection depends on the particular assay and mode of detection. There are several different types of HRP substrates. 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. 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. 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. HRP conjugates are commonly used in indirect assays. ![]() HRP conjugates are used as reporters in a number of common techniques including western blotting, enzyme-linked immunosorbent assays (ELISAs), and microscopy. 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. 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. ![]()
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