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 beta-glucuronidase from complex cell homogenates or partially purified protein fractions is presented. Dual fluorescence is achieved by using the green emitting fluorogenic substrate ELF 97 beta-D-glucuronide to detect beta-glucuronidase activity, followed by the red emitting SYPRO Ruby protein gel stain or SYPRO Ruby IEF gel stain to detect the remaining proteins in the electrophoretic profile. Both ELF 97 alcohol, the highly fluorescent hydrolytic product generated from the enzyme substrate, and the SYPRO Ruby total protein stains are maximally excited by ultraviolet illumination. ELF 97 alcohol emits maximally at 525 nm while the SYPRO Ruby dyes emit maximally at 610 nm. Since ELF 97 beta-glucuronide is a precipitating substrate, it allows precise localization of beta-glucuronidase activity with minimal band diffusion. The staining method is simple and direct, without the requirement for ancillary coupling reactions. Dichromatic protein detection is demonstrated after sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis, carrier ampholyte-mediated isoelectric focusing or two-dimensional gel electrophoresis.
Kemper
C
C
Molecular Probes, Inc, Eugene, OR 97402, USA.
Steinberg
T H
TH
Jones
L
L
Patton
W F
WF
D004586:Electrophoresis / Q000379:methods*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005456:Fluorescent Dyes; D005965:Glucuronates / Q000378:metabolism; D005966:Glucuronidase / Q000032:analysis*; D007525:Isoelectric Focusing / Q000379:methods; D011506:Proteins / Q000032:analysis*; D013050:Spectrometry, Fluorescence / Q000379:methods*
C106153:ELF-97 glucuronide; D005456:Fluorescent Dyes; D005965:Glucuronates; D011506:Proteins; D005966:Glucuronidase