How to use peroxidase and its substrate in biochemical detection
Peroxidase POD is one of the most commonly used enzyme preparations in the field of in vitro diagnostics. Many biochemical tests such as Trinder's reagent enzymatic detection, ELISA enzyme-linked immunoassay, luminol chemiluminescence immunoassay, etc., these related substrates will be Use POD (or HRP) as a catalytic enzyme preparation.
Introduction to Peroxidase
The commercially available peroxidase is usually horseradish peroxidase (HRP) extracted from horseradish with a molecular weight of about 40,000. It is a heme-containing protein with an isoelectric point of 7.2; it is easily soluble Water, the solubility is 5% (W/V), the solution is brownish-red and transparent; it is also soluble in ammonium sulfate solution with saturation below 0.58, but insoluble above 0.62 saturation. The optimal pH of the enzyme is 7.0 (for guaiacol as a hydrogen donor). HRP remains stable for several weeks at room temperature, and is stable within 15 minutes after heating to 63°C.
New Trinder’s Reagent for Peroxidase and Chromogen Substrate
New Trinder's reagents such as TOOS, MAOS, ADPS, etc. are important chromogen substrates for peroxidase. In the Trinder reaction, POD is the catalyst, hydrogen peroxide is the oxidant, and Trinder's reagent is coupled with 4-AAP as the hydrogen donor. Reducing agent, and finally produce red quinone imine substance as a color product. The amount of colored product is directly proportional to the content of hydrogen peroxide, and the amount of colored product can be measured by a spectrophotometer. This reaction can be used for the detection of indicators that can generate hydrogen peroxide, such as the detection of biochemical items such as blood lipids, blood sugar, cholesterol, and creatinine.
Peroxidase and substrate TMB
TMB is tetramethylbenzidine, which is also a chromogenic substrate for POD, and is widely used in ELISA kits. The oxidative coloring principle of benzidine is similar to Trinder’s reagent, but it is mutagenic (carcinogenic) to cells. TMB is an improved product on the basis of benzidine, eliminating the mutagenic form.
Peroxidase and Luminol
In addition to chromogen substrates, peroxidase also has luminescent substrates. Luminol, isoluminol, ABEI, etc. are all commonly used luminescent substrates for POD. Luminol can be catalyzed by hydrogen peroxide to produce chemiluminescence in the presence of alkaline and POD, which can be measured with a photometer.
There are other types of peroxidase substrates, but these are more widely used. Relatively speaking, the luminescent substrate luminol has higher sensitivity. In addition, luminol can detect traces of blood is better than the blood containing hemoglobin. Desheng can provide various chromogen substrates, luminescent substrates, and related enzyme preparations and buffers for the peroxidase detection system.
HEPES, as a zwitterionic buffer, increases the osmotic pressure of the cell culture system by increasing the concentration of solution ions, maintaining normal cell morphology and function, and improving cell survival rate. Widely used in cell culture, especially under specific conditions such as tumor cell culture, it is crucial to maintain cell growth and function.