ELISA TMB Substrate for HRP

G443250 ml


Description3,3'5,5'-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.
ApplicationsSuitable for kinetic and endpoint assays involving Horseradish Peroxidase (HRP).
Protocol Overview1. 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 ConditionsThe 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 10e-7mol/l of iron or copper salts, otherwise, unreacted TMB will be converted non-enzymatically to the diimine.
Unit quantity250 ml
Storage ConditionStore 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.

Supporting Protocol





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          • Löffler, T et al. "Decreased Plasma Aβ in Hyperlipidemic APPSL Transgenic Mice Is Associated with BBB Dysfunction" Frontiers in Neuroscience 232 (10): (2016). DOI: 10.3389/fnins.2016.00232. Application: ELISA.