TMB Slow Kinetic 1-Component HRP Microwell Substrate (SUBK) is suitable for use in all ELISAs where the target detection level is in the µg-ng/ mL range and horseradish peroxidase (HRP) is the conjugated detection enzyme. For assays requiring greater sensitivity, use TMB substrates SUBT (catalog #6276) or SUBS (catalog #6275). SUBK should not be used for membrane or immunohistochemical applications.

SUBK is a one-component, ready-to-use formulation containing 3,3’,5,5’-tetramethylbenzidine (TMB) in a mildly acidic buffer that does not contain aprotic solvents. TMB substrate is oxidized by the peroxidase enzyme to yield a soluble blue-green reaction product, which can be read at 370 nm or 620-650 nm. In endpoint assays, the reaction can be stopped by adding equal volumes of Stop Solution for TMB Microwell Substrates (STOPT, catalog #6282). Addition of STOPT changes the chromagen color from blue-green to yellow, where it can be read at 450 nm, and concurrently stabilizes the yellow TMB product for one hour. Stopping the reaction will increase the sample absorbance value up to 3-fold. To avoid overdeveloping the TMB substrate reaction, the blue-green reaction product should be periodically monitored on an ELISA plate reader using 620-650 nm absorbance filter settings. When OD values reach approximately 0.7 units, the reaction should be stopped using STOPT.

For best results, the absorbance should be monitored and read before values exceed 2.5 OD units. The substrate should not be diluted. The intensity of the reaction can be reduced by further dilution of the antibodies/conjugates used in the assay or by shortening the incubation time.