1-Step™ TMB ELISA Substrate Solutions

TMB (3,3',5,5' tetramethylbenzidine) is a chromagen that yields a blue color when oxidized with hydrogen peroxide (catalyzed by HRP) with major absorbances at 370 nm and 652 nm. The color then changes to yellow (measured at 450 nm) upon addition of sulfuric or phosphoric acid to stop the reaction. TMB is very sensitive and more quickly oxidized than other HRP substrates, resulting in faster color development. Unlike other commercially available substrates, these products contain no DMF or DMSO.

There are three formulations that differ primarily in their sensitivities. Slow TMB is intermediate in sensitivity, ideal for kinetic readings. The sensitivity of the Turbo TMB compares to that of OPD used at approximately 1 mg/mL. Ultra TMB-ELISA produces the highest signal-to-noise ratio and sensitivity in the picogram range.

Thermo Scientific Pierce 1-Step TMB ELISA Substrates detects horseradish peroxidase (HRP) activity, yielding a blue color (Amax = 370 nm and 652 nm) that changes to yellow (Amax = 450 nm) upon addition of a sulfuric or phosphoric acid stop solution.

Features of 1-Step TMB ELISA Substrates:
• Available in traditional or ready-to-use formulations
• No additional reagents or filtering required
• Non carcinogenic
• Various levels of sensitivity and development speed to suit any assay
• No DMF or DMSO present in the reagents

TMB is the most popular chromogenic substrate for HRP detection in ELISA and is available in several formats. The Pierce 1-Step TMB Substrates are one-component substrates that require no preparation before use. Unlike other commercially available substrates, these products contain no DMF or DMSO.

Thermo Scientific Pierce 1-Step Ultra TMB yields the greatest sensitivity among the TMB substrates, followed by 1-Step Turbo TMB and 1-Step Slow TMB. The sensitivity of the 1-Step Turbo TMB is similar to OPD-based substrates formulated at approximately 1 mg/mL. 1-Step Slow TMB has intermediate sensitivity—more sensitive than ABTS, but less sensitive than o-phenylenediamine dihydrochloride (OPD) or 1-Step Turbo TMB. The 1-Step Slow TMB is an ideal substrate for kinetic studies.

TMB (3,3',5,5'-tetramethylbenzidine) is a chromogen that yields a blue color when oxidized, typically as a result of oxygen radicals produced by the hydrolysis of hydrogen peroxide by HRP. For kinetic or non-stopped ELISA assays, the TMB chromogen has maximal absorbances at 370 nm and 652 nm. The color then changes to yellow with the addition of sulfuric or phosphoric acid with maximum absorbance at 450 nm. A green reaction product may result from partial conversion to the yellow product from the blue intermediate. TMB is very sensitive ELISA substrate and is more quickly oxidized than other HRP substrates, resulting in faster color development.