TMB

3,3',5,5'-Tetramethylbenzidine

CAS 54827-17-7

CAS: 54827-17-7
Reading: 450 nm after an acid stop
Form: Raw material

TMB is the chromogen. Hydrogen peroxide and the stop acid are part of the laboratory method. The powder is light sensitive.

What it is

TMB (3,3',5,5'-tetramethylbenzidine) is a chromogenic hydrogen donor for horseradish peroxidase. HRP and hydrogen peroxide oxidize it to a blue product read near 650 nm; adding acid converts it to a yellow diimine read at 450 nm with higher sensitivity. It was introduced as a less hazardous alternative to benzidine.

Where it is used

ELISA and IVD immunoassays: soluble substrate, stopped with dilute sulfuric or phosphoric acid (often 0.5–2 M H₂SO₄).
Western blot and dot blot, using precipitating TMB formulations.
Immunohistochemistry and other HRP-based detections.

Handling tips

TMB base is poorly soluble in water. Make a stock in DMSO or ethanol and dilute into citrate or acetate buffer with H₂O₂ just before use.
Protect powder and working solutions from light and metal contamination; a blue tint before use indicates oxidation.
Sodium azide inhibits HRP, so keep it out of wash and substrate buffers.

What to specify when ordering

TMB free base or TMB dihydrochloride (more water-soluble).
Powder, or ready one-component substrate solution.
Purity and pack size. BSA, PBS and Tween 20 can be inquired together.

Properties and storage

Formula: C16H20N2
Molecular weight: 240.34
Appearance: White to pale yellow crystalline powder, light sensitive
Solubility: Soluble in DMSO and ethanol; poorly soluble in water
Storage: 2–8 °C, dry, dark
Typical packs: 1 g, 5 g, 25 g

Typical literature values. The batch COA / SDS governs.

Before you inquire

Is this a ready substrate solution?

It is the TMB raw material. Name powder, solution or dihydrochloride on the inquiry.

Why read at 450 nm after stopping?

The acid turns the blue charge-transfer complex into a yellow diimine. It is stable for a short time and absorbs more strongly, so plates are read soon after the stop.

TMB or ABTS?

TMB is generally more sensitive. ABTS develops more slowly and gives a green product read near 405–414 nm. It is also on the stains page.

Pack sizes and catalogue numbers

CTH10142-100G

Pack: 100 g
Lead time: On request

Inquire

Prices on request; lead time confirmed on the quotation.

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For research or industrial use only. Not for human or animal diagnosis or treatment, and not for drug or food use. Grade, pack size and lead time follow the supplier batch COA / SDS. Figures on this site are general industry references, not a factory specification.

TMB