TMB 1-Component HRP Microwell Substrate is a ready-to-use solution containing 3,3′,5,5′-tetramethylbenzidine in a mildly acidic buffer that does not contain aprotic solvents. This substrate is ideal for use in all ELISAs where the target detection level is in the ng-pg/mL range and horseradish peroxidase (HRP) is the conjugated detection enzyme.
- Allow product to equilibrate to room temperature (25°C) prior to use.
- Add 100 µL to each well. When reacting with peroxidase, a soluble blue reaction product develops that can be read at 370 nm or in a range of 620-650 nm.
- The reaction can be stopped using an appropriate stop solution, such as Stop Solution for TMB Substrates (STOPT), for endpoint assays. STOPT changes the chromogen from blue to yellow and the absorbance to 450 nm. For best results, sample absorbance should be monitored and read before values exceed 2.5 OD units. STOPT can increase the absorbance up to 3-fold, so the reaction should be stopped when absorbance values reach approximately 0.7 OD units.