Chemical substrates can be used for various applications, including immunohistochemistry, immunoassay, Western blotting, and functional analysis. Western blotting is a commonly used tool to identify and quantify specific proteins in complex mixtures. This technique makes it possible to indirectly detect protein samples immobilized on nitrocellulose or polyvinylidene difluoride (PVDF) membranes. To detect the target protein, a primary antibody against the target protein is applied to the membrane as a probe, and the enzyme-conjugated secondary antibody binds to the primary antibody. In western blot, the two most frequently used enzymes that bind secondary antibodies are horseradish peroxidase (HRP) and alkaline phosphatase (AP). The substrates react with enzyme labels to visualize or measure target proteins through visible color, fluorescence or chemiluminescence. The most significant advances in Western blotting are highly sensitive enhanced chemiluminescent (ECL) substrates, imaging systems, and a variety of photostable fluorophores. The ECL substrate can detect proteins at low femtogram levels with high signal-to-noise ratios.
Chemiluminescence is a chemical reaction in which energy is released in the form of light. The widespread use of extremely sensitive chemiluminescence substrates has virtually eliminated the use of radioisotope-labeled probes. The most popular chemiluminescent HRP substrates for Western blotting are luminol-based. In the presence of HRP and peroxide buffer, luminol oxidizes and forms an excited state product, which emits light as it decays to the ground state. Light emission occurs only during the enzyme-substrate reaction, so signal output ceases once the substrate in proximity to the enzyme is depleted. In contrast, colorimetric substrates, such as TMB, produce precipitates that are still visible on the membrane even after the reaction has terminated.
Amerigo Scientific offers value-priced substrates for western blot detection of enzyme conjugates, including chemiluminescent or chromogenic precipitation substrates.
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