3,3′,5,5′-Tetramethylbenzidine (TMB) is a chromogenic substrate suitable for use in ELISA procedures, which utilize horseradish peroxidase conjugates. This substrate produces a soluble end product that is blue in color and can be read spectrophotometrically at 370 or 655 nm. The reaction maybe stopped with acid, resulting in a yellow solution that is read at 450 nm. This product is supplied as a ready-to-use peroxidase substrate containing TMB in a mildly acidic buffer. Prior to reaction with peroxidase, the substrate should be a colorless to light bluish-green solution. The substrate system develops a blue reaction product when reacted with peroxidase in microwell applications (e.g. ELISA assays). For end-point assays, acid can be used to stop the reaction, to yield a yellow end product. Since this substrate produces a soluble reaction product, it is not recommended for histochemistry or blotting.
Following reaction with peroxidase, a blue reaction product forms that may be read at 370 nm or between 620 and 655 nm. For end-point assays, the reaction can be stopped by the addition of a volume of 1 M or 2 M HCl, or 0.5 M H2SO4, equal to the volume of the substrate reaction in the well. The resulting yellow end product, which is stable for at least one hour, can then be read at 450 nm. Dilution of the substrate is not recommended. To reduce the intensity of a reaction, it is recommended that the antibodies or conjugates be diluted.
TMB is a combination of buffer, chromogen and H₂O₂, as a substrate, in one convenient single component reagent.
Store at 4ºC. Protect from light.
For laboratory use only. Not for any clinical, therapeutic, or diagnostic use in humans or animals. Not for animal or human consumption.
3-3',5,5'-tetramethylbenzidine (TMB) is a soluble chromogen substrate for horseradish peroxidase detection systems. It is recommended for ELISA procedures.