Microfabricated channel array electrophoresis for characterization and screening of enzymes using RGS-G protein interactions as a model system.
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 x 7.62 cm(2) 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.
Pei
Jian
J
Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109-1055, USA.
Dishinger
John F
JF
Roman
David L
DL
Rungwanitcha
Chetwana
C
Neubig
Richard R
RR
Kennedy
Robert T
RT
D001896:Boron Compounds / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods*; D020558:GTP Phosphohydrolases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000032:analysis* / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000737:chemistry; D006153:Guanosine Diphosphate / Q000737:chemistry / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods*; D020710:RGS Proteins / Q000032:analysis* / Q000378:metabolism; D012235:Rhodamines / Q000737:chemistry
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D020710:RGS Proteins; D012235:Rhodamines; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins