Substrate Reagent A should be clear and colorless. Substrate Reagent B should be colorless to a very light amber. TMB Substrate is the mixture of equal volumes of TMB Substrate Reagent A with TMB Substrate Reagent B. For example, for one plate, mix 6 ml Substrate Reagent A with 6 ml of Substrate Reagent B in a clean container. Mix immediately before use. After mixing the Reagents together, TMB substrate working solution should be colorless or very faint blue.
Application Notes:
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.
Application References:
1. Kim, MS., et al. 2008. J Biochem. 143:497. PubMed
Description:
The TMB Substrate Reagent Set contains Reagent A and Reagent B. 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.
*These products may be covered by one or more Limited Use Label Licenses (see the BioLegend Catalog or our website, www.biolegend.com/ordering/). BioLegend products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without written approval of BioLegend. By use of these products you accept the terms and conditions of all applicable Limited Use Label Licenses. Unless otherwise indicated, these products are for research use only and are not intended for human or animal diagnostic, therapeutic or commercial use.
