Applications: Suitable for kinetic and endpoint assays involving Horseradish Peroxidase (HRP).
Protocol Overview: 1. Complete all required incubations with antibodies, probes and HRP labeled reagents.
2. Wash plate, at least 4 times, with phosphate buffered saline or Tris buffered saline containing 0.1% Tween-20.
3. After the final wash, shake and blot all residual buffers from plate wells.
4. Add 0.1ml of TMB Substrate for HRP to the appropriate wells and incubate 5-30 minutes.
5. (Optional Higher Sensitivity Reaction Stop) Highest sensitivity of detection is achieved by stopping the TMBHK reaction with an equal volume of 0.1ml of 0.3M of sulphuric acid. Read the more highly absorbing yellow chromogen in the stopped reaction at 450nm.
6. Assay kinetics may be monitored at 650nm as a function of time. The reaction may be stopped to preserve the blue chromogen using an equal volume of 0.1ml of 0.1% sodium fluoride or 0.15% sodium dodecyl sulfate. Measure the blue chromogen in the stopped reaction at 650nm.
Shipping Conditions: The reaction time will depend upon the activity of the HRP probe. If color develops too briskly, zero order kinetics will not prevail. Dilution of probe, antibody or HRP-labelled reagent may be required and optimized by the end user for their specific application. In addition, time, reagent volume and temperature variations may require standardisation by the user. Reagent grade water must contain less than 107 mol/l of iron or copper salts, otherwise, unreacted TMB will be converted non-enzymatically to the diimine.
Storage Condition: Store at 2-4°C and then warm up to assay temperature prior to use. ELISA TMB Substrate for HRP can also be stored at room temperature for up to 6 months. Prevent exposure to direct sunlight. Discard if solution turns blue or turbid.