The product is Factory Supply 3,3′,5,5′-tetramethylbenzidine dihydrOchlOride hydrate. It has a purity level of 98% and its CAS number is 207738-08-7. This product is provided at the best price by the factory. 3,3′,5,5′-tetramethylbenzidine (TMB) dihydrochloride hydrate is a chromogenic substrate commonly used in enzyme immunoassays (EIAs) such as ELISA. It is a white to off - white powder in appearance when in a pure state. The solubility of this compound is an important parameter. It is soluble in water and some polar organic solvents to a certain extent, which makes it suitable for use in aqueous - based biological assays.
This product is mainly used in the field of life sciences, especially in immunoassays. In enzyme - linked immunosorbent assays (ELISA), TMB dihydrochloride hydrate serves as a chromogenic substrate for horseradish peroxidase (HRP). When HRP catalyzes the reaction in the presence of hydrogen peroxide, TMB is oxidized from a colorless state to a blue product. After the addition of an acidic stop solution, the blue color turns yellow, and the absorbance can be measured at a specific wavelength (usually 450 nm). This allows for the quantitative detection of antigens or antibodies in the sample. Besides ELISA, it can also be used in immunohistochemistry, where it helps to visualize the localization of specific proteins in tissue sections. It is also useful in some enzyme - based biosensors for detecting various analytes in biological and environmental samples.
When using 3,3′,5,5′-tetramethylbenzidine dihydrochloride hydrate in ELISA, first, prepare the TMB substrate solution according to the manufacturer's instructions. Usually, it involves dissolving the powder in a suitable buffer solution. Ensure that the solution is well - mixed and free of any undissolved particles. Before adding the TMB substrate to the ELISA plate, make sure that the plate has been properly coated with the capture antibody and blocked to prevent non - specific binding. Then, add the appropriate amount of sample and detection antibody to the wells. After incubation and washing steps, add the TMB substrate solution to each well. Incubate the plate in the dark for a specific period (usually 15 - 30 minutes) to allow the color reaction to occur. Finally, add the stop solution to terminate the reaction and measure the absorbance using a microplate reader. In immunohistochemistry, the TMB solution is applied to the tissue sections after appropriate antigen retrieval and antibody incubation steps. The staining time and conditions may vary depending on the specific tissue and target antigen.
In a research project focused on detecting a specific virus in clinical samples, an ELISA assay was developed using 3,3′,5,5′-tetramethylbenzidine dihydrochloride hydrate as the chromogenic substrate. The researchers first coated the ELISA plate with a monoclonal antibody specific to the virus. After blocking the plate, they added the patient serum samples. A secondary antibody conjugated with HRP was then added. After a series of washing steps, the TMB substrate solution was added. The blue color development was clearly visible in the positive samples, and after adding the stop solution, the yellow color intensity was measured. The results showed a strong correlation between the absorbance values and the presence of the virus in the samples, indicating the high sensitivity and specificity of the assay. In another case, in an immunohistochemistry study of a tumor tissue, TMB dihydrochloride hydrate was used to visualize the expression of a tumor - associated protein. The tissue sections were first treated with an antigen - retrieval buffer, followed by incubation with a primary antibody against the protein and then a secondary antibody conjugated with HRP. When the TMB solution was applied, the positive staining areas in the tissue sections were clearly distinguishable, helping the researchers to understand the distribution and expression level of the protein in the tumor.
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