Microfabricated Channel Array Electrophoresis for Characterization and Screening of Enzymes Using RGS−G Protein Interactions as a Model System
- 9 May 2008
- journal article
- research article
- Published by American Chemical Society (ACS) in Analytical Chemistry
- Vol. 80 (13), 5225-5231
- https://doi.org/10.1021/ac800553g
Abstract
A microfluidic chip consisting of parallel channels designed for rapid electrophoretic enzyme assays was developed. Radial arrangement of channels and a common waste channel allowed chips with 16 and 36 electrophoresis units to be fabricated on a 7.62 × 7.62 cm2 glass substrate. Fluorescence detection was achieved using a Xe arc lamp source and commercial charge-coupled device (CCD) camera to image migrating analyte zones in individual channels. Chip performance was evaluated by performing electrophoretic assays for G protein GTPase activity on chip using BODIPY−GTP as enzyme substrate. A 16-channel design proved to be useful in extracting kinetic information by allowing serial electrophoretic assays from 16 different enzyme reaction mixtures at 20 s intervals in parallel. This system was used to rapidly determine enzyme concentrations, optimal enzymatic reaction conditions, and Michaelis−Menten constants. A chip with 36 channels was used for screening for modulators of the G protein−RGS protein interaction by assaying the amount of product formed in enzyme reaction mixtures that contained test compounds. Thirty-six electrophoretic assays were performed in 30 s suggesting the potential throughput up to 4320 assays/h with appropriate sample handling procedures. Both designs showed excellent reproducibility of peak migration time and peak area. Relative standard deviations of normalized peak area of enzymatic product BODIPY−GDP were 5% and 11%, respectively, in the 16- and 36-channel designs.Keywords
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