Sensitive, soluble colorimetric HRP substrates for protein detection in ELISA

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

Highlights of TBM 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 reagent

About TMB ELISA Substrates:

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 370nm and 652nm^1,2. The color then changes to yellow with the addition of sulfuric or phosphoric acid with maximum absorbance at 450 nm^1,2. 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 development³.

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

For a comparison of all Thermo Scientific Pierce ELISA Substrates, see the Guide to ELISA Substrates

References:

1. Josephy, P.D., et al. (1982). J. Biol. Chem. 257(7), 3669-3675.
2. Josephy, P.D., et al. (1983). J. Biol. Chem. 258(9), 5561-5569.
3. Liem, H.H., et al. (1979). Anal. Biochem. 98, 388-393.
4. Thomas, P.E., et al. (1976). Anal. Biochem. 75, 168-176.
5. Hong, P.W., et al. (2002). J. Virol. 76, 12855-12865.
6. Tek, V. and Zolkiewski, M. (2002). Protein Sci. 11, 1192-1198.
7. Wu, S.-C. and Wong, S.-L. (2002). Appl. Envir. Microbiol. 68, 1102-1108.
8. Su, S.V., et al. (2004). J. Biol. Chem. 279(18), 19122-19132.
9. Weimer, B.C., et al. (2001). Appl. Envir. Microbiol. 67, 1300-1307.
10. Murphy, M.B., et al. (2003). Nucleic Acids Res. 31, e110.

Related Links
Selection Guide: Enzyme Substrates for ELISA
Spike-and-recovery and linearity-of-dilution assessment

Related Products
Precipitating TMB Substrates for Western blotting
ABTS
OPD (o-Phenylenediamine)
PNPP Substrates
Coated and Uncoated Microwell Plates
ELISA Products