Sie befinden Sich nicht im Netzwerk der Universität Paderborn. Der Zugriff auf elektronische Ressourcen ist gegebenenfalls nur via VPN oder Shibboleth (DFN-AAI) möglich. mehr Informationen...
HTRF Kinase Assay Development and Methods in Inhibitor Characterization
Ist Teil von
Methods in molecular biology (Clifton, N.J.), 2016, Vol.1360, p.1
Ort / Verlag
United States
Erscheinungsjahr
2016
Quelle
MEDLINE
Beschreibungen/Notizen
Due to their important roles in cellular signaling and their dysfunctions being linked to diseases, kinases have become a class of proteins being actively pursued as potential drug targets. Biochemical assays for kinases have been developed in various formats to facilitate inhibitor screening and selectivity profiling. Here, we focus on one such technology: homogeneous time-resolved fluorescence (HTRF). In this chapter, we describe the methods of developing an HTRF kinase assay using mutant EGFR enzyme as an example. We show how to determine the kinetic parameter of the enzyme (ATP K m), as well as how to study the inhibitor mechanism of action (MoA) exemplified by inhibitors of different MoAs. All methods described here can be readily applied to other kinases with minor modifications.