Thermo Scientific Pierce ONPG is the preferred colorimetric substrate for ELISA applications involving beta-galactosidase (b-Gal) as the reporter enzyme.

When using beta-Galactosidase as the label for proteins in ELISA studies, a wide variety of substrates are available, including: o-Nitrophenyl-beta-D-galactopyranosidase (ONPG), Naphthol-AS-Bl-beta-D-galactopyranosidase (Nap-Gal), and 4-Methyl-umbelliferyl-beta-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 beta-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.