Thermo Scientific Pierce ONPG is the preferred colorimetric substrate for ELISA applications involving beta-galactosidase (b-Gal) as the reporter enzyme.
When using ß-Galactosidase as the label for proteins in ELISA studies, a wide variety of substrates are available, including: o-Nitrophenyl-ß-D-galactopyranosidase (ONPG), Naphthol-AS-Bl-ß-D-galactopyranosidase (Nap-Gal), and 4-Methyl-umbelliferyl-ß-D-galactopyranosidase (MUm-Gal). However, it is important to choose a substrate with adequate solubility, that uses readily available equipment, and that gives a significant reading over the background. ONPG is a superior ß-Galactosidase substrate option.(1) The product formed is completely soluble and has a high extinction coefficient at 405nm. The substrate yields a yellow product that is easily detectable in the visual range after stopping the reaction with 1M Sodium Carbonate.
- Craven, G.R., Steers, Jr., E., and Anfinsen, C.B.,(1965). Purification, composition and molecular weight of the ß-Galactosidase of Escherichia coli K12. J. Biol. Chem. 240, 2468-2477.