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TMB (3,3′,5,5′-tetramethylbenzidine) is a chromogenic substrate for the HRP (horseradish peroxidase) detection enzyme, supplied as a ready-to-use solution for colorimetric ELISA. HRP oxidizes TMB to produce a soluble blue reaction product; when the reaction is stopped, the chromophore shifts to yellow and is read at 450 nm. TMB is widely used because it offers high sensitivity, low background and a measurable absorbance endpoint.
How Does TMB Work?
TMB acts as an electron donor. Bound HRP catalyzes the reaction between hydrogen peroxide (H₂O₂) and TMB, oxidizing TMB into a colored reaction product whose absorbance is proportional to the amount of HRP — and therefore to the amount of analyte captured in the assay. Adding a stop solution prevents further color development and shifts the chromophore to yellow, which you read at 450 nm.
Chemistry at a glance
TMB (3,3′,5,5′-tetramethylbenzidine) is oxidized in the presence of H₂O₂ to a blue charge-transfer complex that absorbs around 370 nm and 650 nm. Stopping the reaction with acid protonates this complex into a yellow diimine that absorbs at 450 nm. Because the developed color is directly proportional to enzyme (and therefore analyte) level, the absorbance can be read quantitatively — and it is this soluble, bleachable product that makes TMB convenient for microwell assays compared with precipitating substrates.
Where Is TMB Used?
TMB provides flexible colorimetric detection across immunoassay platforms — ELISA, ELISpot, Western blot and protein microarrays — wherever HRP is the reporter enzyme:
ELISA — sandwich, direct, indirect and competitive formats
Immunoassay kit development — signal readout for HRP conjugates
Cell-based and enzyme assays that use HRP as the reporter
TMB vs Other Enzyme Substrates
| Substrate | Readout | Typical Use | Note |
|---|---|---|---|
| TMB | Blue → yellow, 450/650 nm | Soluble ELISA (HRP) | High sensitivity, low background |
| OPD | Orange-brown, 492 nm | HRP | Classic choice |
| ABTS | Green, 405 nm | HRP | Stable endpoint |
| pNPP | Yellow, 405 nm | Alkaline phosphatase (AP) | AP substrate |
| BCIP/NBT | Purple/blue precipitate | Membrane / Western blot | Precipitating substrate |
Durastab™ TMB Substrate Options
Durastab™ offers four one-component TMB substrates — High Sensitive, One Component, Board Range and Slow Plus — covering different kinetics and detection ranges, plus freeze-thaw resistant variants (suffix -F). All are ISO9001-certified for consistent batch-to-batch performance.
Quick Spec Highlights
| Parameter | Typical Value |
|---|---|
| Absorbance read | 450 nm (stopped) / 650 nm (unstopped) |
| Storage | 2–8 °C; protect from light |
| Shelf life | Up to 4 years (by formulation) |
| Stability | >1 year open-vial at 2–8 °C |
| Formats | 100 mL, 1 L, 5 L, 10 L, bulk |
