Utilization of luminescent technology to develop a kinase assay: Cdk4 as a model system.
Protocols to assess kinase activity generally include radioactive methods, fluorescent polarization technology and the use of specific antibodies. Here, a simple, effective, non radioactive method to measure kinase activity of immunoprecipitated proteins is described. Cdk4, a cell cycle dependent enzyme, was immunoprecipitated from whole cell extracts and used in kinase reactions. This system has been developed taking advantage of the kinase-Glo reagent (Promega), based on ATP depletion technology, but with a wider range of applications. The original aim of the commercial kit is the evaluation of kinase activity of highly purified enzymes, while this system enabled the evaluation of native kinases, retrieved by immunoprecipitation. This method was highly homogeneous and did not require any kind of separation or purification as well. Moreover, it was suitable for basic research and may be useful for low-medium throughput pharmaceutical screening of chemical libraries.
Tagliati
Federico
F
Section of Endocrinology, Department of Biomedical Sciences and Advanced Therapies, University of Ferrara, Via Savonarola 9, 44100 Ferrara, Italy.
Bottoni
Arianna
A
Bosetti
Alessandro
A
Zatelli
Maria Chiara
MC
degli Uberti
Ettore C
EC
D000255:Adenosine Triphosphate / Q000737:chemistry; D001671:Biochemistry / Q000379:methods*; D002453:Cell Cycle; D002626:Chemistry, Pharmaceutical / Q000379:methods; D051358:Cyclin-Dependent Kinase 4 / Q000032:analysis* / Q000302:isolation & purification*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004345:Drug Industry / Q000379:methods; D004798:Enzymes / Q000032:analysis; D016231:Fluorescence Polarization Immunoassay / Q000379:methods; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D007211:Indoles / Q000032:analysis; D049449:Luminescence; D015233:Models, Statistical; D010091:Oximes / Q000032:analysis; D010766:Phosphorylation; D013678:Technology, Pharmaceutical / Q000379:methods*; D013997:Time Factors
D004798:Enzymes; D007211:Indoles; D010091:Oximes; C474021:indirubin-3'-monoxime; D000255:Adenosine Triphosphate; D051358:Cyclin-Dependent Kinase 4