The product named "High Quality 3,3′,5,5′-Tetramethylbenzidine Dihydrochloride With Best Price" is a high - quality chemical compound. 3,3′,5,5′-Tetramethylbenzidine Dihydrochloride, often abbreviated as TMB-HCl, is a chromogenic substrate used in ELISA (Enzyme - Linked Immunosorbent Assay) and other immunological assays.
Basic parameters:
It usually appears as a white to off - white powder. The molecular formula of 3,3′,5,5′-Tetramethylbenzidine Dihydrochloride is C₁₆H₂₀N₂·2HCl, with a molecular weight of approximately 329.27 g/mol. It has good solubility in some organic solvents and aqueous solutions under specific conditions, which makes it convenient for different experimental setups.
1. **ELISA assays**: In ELISA, it serves as a substrate for horseradish peroxidase (HRP). When HRP reacts with TMB-HCl in the presence of hydrogen peroxide, a color change occurs. The intensity of the color can be measured spectrophotometrically, which is proportional to the amount of antigen or antibody in the sample. This makes it a crucial reagent for detecting and quantifying various biomolecules such as proteins, hormones, and antibodies in biological samples.
2. **Immunohistochemistry**: It can be used in immunohistochemical staining procedures. By visualizing the binding of specific antibodies to tissue sections, TMB-HCl helps researchers identify the presence and localization of target antigens in cells and tissues. This is valuable in pathology research, cancer diagnosis, and understanding the distribution of proteins in normal and diseased tissues.
3. **Other enzymatic assays**: Besides HRP, it may also be used in other enzymatic reactions where a chromogenic substrate is required to monitor the enzyme activity. This broadens its application scope in the field of biochemistry and molecular biology.
1. **Preparation of working solution**: First, dissolve the appropriate amount of 3,3′,5,5′-Tetramethylbenzidine Dihydrochloride in a suitable buffer, usually a phosphate - citrate buffer. The concentration of the substrate in the working solution may vary depending on the specific assay requirements, typically in the range of 0.1 - 1 mg/mL. Then, add a small amount of hydrogen peroxide as the co - substrate to initiate the enzymatic reaction.
2. **Assay procedure**: In an ELISA, after the sample has been incubated with the appropriate antibodies and washed to remove unbound substances, the TMB - HCl working solution is added to the wells of the microplate. Incubate the plate at room temperature for a specified period, usually 15 - 30 minutes. During this time, the HRP in the bound antibody - antigen complex catalyzes the reaction between TMB - HCl and hydrogen peroxide, resulting in a color change from colorless to blue.
3. **Stopping the reaction**: After the desired incubation time, the reaction can be stopped by adding a stop solution, usually 2M sulfuric acid. The color then changes from blue to yellow, and the absorbance can be measured at 450 nm using a microplate reader.
Case 1: Cancer biomarker detection
A research team was studying a potential cancer biomarker in blood samples. They used an ELISA kit with 3,3′,5,5′-Tetramethylbenzidine Dihydrochloride as the substrate. First, they coated the microplate wells with capture antibodies specific to the biomarker. After adding the patient's blood samples and incubating, they added detection antibodies labeled with HRP. Then, the TMB - HCl working solution was added. The color development was clearly visible, and by comparing the absorbance values with a standard curve, they were able to accurately quantify the biomarker level in each sample. This helped in early cancer diagnosis and monitoring the progression of the disease.
Case 2: Hormone quantification in animal studies
In an animal endocrinology study, researchers wanted to measure the levels of a particular hormone in the serum of experimental animals. They used an ELISA protocol with TMB - HCl. The detection was highly sensitive, allowing them to detect even small changes in hormone levels over time. This data was crucial for understanding the hormonal regulation mechanisms in the animals under different experimental conditions.
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