The TMB Substrate Reagent Set contains 100 mL both Substrate Reagent A and Substrate Reagent B, sufficient for twenty 96-well plates
Warm to room temperature prior to use. To prepare a working solution, mix equal volumes of Substrate Reagent A and Substrate Reagent B. Prepare only the amount needed for each assay run. After wells are completely washed, to remove unbound antibody and HRP, add 100 μl to 200 μl substrate solution to each well. Discard any remaining working solution after use. For endpoint assays, add 50 μl to 100 μl of Stop Solution (2N H2SO4 or 1M H3PO4), which changes the color from blue to yellow. Read at 450 nm (minus 570 nm for wavelength correction) within 30 minutes of stopping the reaction. For kinetic assays, read the blue color at 620 - 650 nm.
It is formulated with 3, 3', 5, 5' tetramethyl benzidine (TMB). The substrate can be catalyzed with peroxidase to produce a pale blue color which can be read spectrophotometrically at 370 or 620-650 nm. The TMB reaction may be stopped with 2N H2SO4 or 1M H3PO4 (resulting in a yellow color), and read at 450 nm.
