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Description 3,3'5,5'-Tetramethylbenzidine (TMB) is a safe-sensitive substrate used for the detection of Horseradish Peroxidase (HRP) activity. It yields a blue colour (Amax = 652nm) and changes to yellow (Amax = 450nm) upon the addition of either sulfuric or phosphoric acid. ABM’s ELISA TMB Substrate for HRP is a highly sensitive single component reagent that is ready to use for the quantitative detection of HRP bound to a solid phase or in free solution.
Protocol 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 ABM’s TMB Substrate for HRP to appropriate wells and incubate 5-30 minutes. 5. The reaction can be monitored as a function of time for kinetic assays or stopped with 0.1ml of 0.3mol/l of sulphuric acid or 0.1% sodium fluoride and read at 650nm. 6. If reaction needs to be converted to yellow diimine, add 0.1ml of either sulfuric or phosphoric acid and record the absorbance within 5 minutes.
Application Suitable for kinetic and endpoint assays involving Horseradish Peroxidase (HRP).
Storage 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.
Cautions 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 the probe, antibody or HRP labeled reagent may be required. In addition, time, reagent volume and temperature variations may require standardization by the user. Reagent grade water must contain less than 10e-7mol/l of iron or copper salts, otherwise, unreacted TMB will be converted non-enzymatically to the diimine.


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