title: Time-resolved fluorescence resonance energy transfer as a versatile tool in the development of homogeneous cellular kinase assays.
abstract: Homogeneous cellular assays can streamline product detection in the drug discovery process. One commercially available assay employing time-resolved fluorescence resonance energy transfer (TR-FRET) that detects phosphorylated products was used to evaluate inhibitors of the receptor tyrosine kinase AXL in a cell line expressing an AXL-green fluorescent protein fusion protein. This TR-FRET assay was modified to evaluate the phosphorylation state of the AXL family member MER in a cell line expressing MER with a V5 tag by adding a fluorescein-labeled anti-V5 antibody. This homogeneous cellular assay was further modified to evaluate the nonreceptor tyrosine kinase focal adhesion kinase (FAK) in cell lines that expressed an untagged kinase by the inclusion of a commercially available anti-FAK antibody conjugated with an acceptor dye. The methods described here can be further adapted for TR-FRET detection of other cellular kinase activities.
authors.affiliation: Worldwide Discovery Research, Cephalon, Inc., West Chester, Pennsylvania 19380, USA.
authors.forename: Lisa
authors.initials: L
authors.lastname: Saville
authors.forename: Chrysanthe
authors.initials: C
authors.lastname: Spais
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Mason
authors.forename: Mark S
authors.initials: MS
authors.lastname: Albom
authors.forename: Seetha
authors.initials: S
authors.lastname: Murthy
authors.forename: Sheryl L
authors.initials: SL
authors.lastname: Meyer
authors.forename: Mark A
authors.initials: MA
authors.lastname: Ator
authors.forename: Thelma S
authors.initials: TS
authors.lastname: Angeles
authors.forename: Jean
authors.initials: J
authors.lastname: Husten
mesh_terms: D000906:Antibodies / Q000032:analysis / Q000276:immunology; D000888:Antibodies, Anti-Idiotypic / Q000032:analysis / Q000276:immunology; D002460:Cell Line; D004396:Coloring Agents; D018076:DNA, Complementary / Q000235:genetics; D003627:Data Interpretation, Statistical; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D019793:Fluorescein; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000032:analysis / Q000037:antagonists & inhibitors; D006801:Humans; D010770:Phosphotransferases / Q000032:analysis* / Q000037:antagonists & inhibitors*; D016133:Polymerase Chain Reaction; D011518:Proto-Oncogene Proteins / Q000032:analysis / Q000037:antagonists & inhibitors; D020794:Receptor Protein-Tyrosine Kinases / Q000032:analysis / Q000037:antagonists & inhibitors; D000076202:c-Mer Tyrosine Kinase
chemical_list: D000906:Antibodies; D000888:Antibodies, Anti-Idiotypic; D004396:Coloring Agents; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D011518:Proto-Oncogene Proteins; D010770:Phosphotransferases; C097072:MERTK protein, human; D020794:Receptor Protein-Tyrosine Kinases; D000076202:c-Mer Tyrosine Kinase; D051416:Focal Adhesion Protein-Tyrosine Kinases; D019793:Fluorescein