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.

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.

1. Kim MS, et al. 2008. J Biochem. 143:497. PubMed
2. McNally A, et al. 2011. PNAS. 108:7529. PubMed