This one-component water based tetramethylbenzidine (TMB) chromogenic substrate is ideal for color development of horseradish peroxide (HRP) based ELISA systems. It is identical to the TMB substrate supplied in our ELISA kits. For best results, add 100 ul to each well and shake at 300 rpm at room temperature for 2-15 minutes. Quench reaction with 50 ul of 1N (0.5M) H2SO4, mix gently, and read plate at 450 nm. Store at 4C and bring to room temperature before use.