title: Diced electrophoresis gel assay for screening enzymes with specified activities.
abstract: We have established the diced electrophoresis gel (DEG) assay as a proteome-wide screening tool to identify enzymes with activities of interest using turnover-based fluorescent substrates. The method utilizes the combination of native polyacrylamide gel electrophoresis (PAGE) with a multiwell-plate-based fluorometric assay to find protein spots with the specified activity. By developing fluorescent substrates that mimic the structure of neutrophil chemoattractants, we could identify enzymes involved in metabolic inactivation of the chemoattractants.
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Toru
authors.initials: T
authors.lastname: Komatsu
authors.forename: Kenjiro
authors.initials: K
authors.lastname: Hanaoka
authors.forename: Alexander
authors.initials: A
authors.lastname: Adibekian
authors.forename: Kentaro
authors.initials: K
authors.lastname: Yoshioka
authors.forename: Takuya
authors.initials: T
authors.lastname: Terai
authors.forename: Tasuku
authors.initials: T
authors.lastname: Ueno
authors.forename: Mitsuyasu
authors.initials: M
authors.lastname: Kawaguchi
authors.forename: Benjamin F
authors.initials: BF
authors.lastname: Cravatt
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Nagano
mesh_terms: D000180:Acrylic Resins / Q000737:chemistry; D000818:Animals; D002630:Chemotactic Factors; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004798:Enzymes / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry; D004950:Esterases / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D005470:Fluorometry; D005959:Glucosidases / Q000737:chemistry; D057166:High-Throughput Screening Assays; D007525:Isoelectric Focusing; D008099:Liver / Q000737:chemistry / Q000201:enzymology; D013058:Mass Spectrometry; D051379:Mice; D018716:Molecular Mimicry; D009504:Neutrophils / Q000737:chemistry / Q000201:enzymology; D010447:Peptide Hydrolases / Q000737:chemistry; D011506:Proteins / Q000737:chemistry; D000099166:Polyacrylamides
chemical_list: D000180:Acrylic Resins; D002630:Chemotactic Factors; D004798:Enzymes; D004950:Esterases; D005456:Fluorescent Dyes; D005959:Glucosidases; D010447:Peptide Hydrolases; D011506:Proteins; D000099166:Polyacrylamides