Simultaneous, two-color fluorescence detection of total protein profiles and beta-glucuronidase activity in polyacrylamide gel

A dichromatic method for measuring the specific activity of P-glucuronidase from complex cell homogenates or partially purified protein fractions is p resented. Dual fluorescence is achieved by using the green emitting fluorog enic substrate ELF 97 beta -D-glucuronide to detect beta -glucuronidase act ivity, followed by the red emitting SYPRO Ruby protein gel stain or SYPRO R uby IEF gel stain to detect the remaining proteins in the electrophoretic p rofile. Both ELF 97 alcohol, the highly fluorescent hydrolytic product gene rated from the enzyme substrate, and the SYPRO Ruby total protein stains ar e maximally excited by ultraviolet illumination. ELF 97 alcohol emits maxim ally at 525 nm while the SYPRO Ruby dyes emit maximally at 610 nm. Since EL F 97 beta -glucuronide is a precipitating substrate, it allows precise loca lization of beta -glucuronidase activity with minimal band diffusion. The s taining method is simple and direct, without the requirement for ancillary coupling reactions. Dichromatic protein detection is demonstrated after sod ium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis, carrier amphol yte-mediated isoelectric focusing or two-dimensional gel electrophoresis.