Revvity, 62KBD01PEA, HTRF Kinase-GST Discovery Kit

Overview
​The best known kinase inhibitor, Gleevec (Imatinib), binds a non-activated form of Abl. Such inhibitors are therefore more difficult to detect using activity based assays, so it is worthwhile to set up kinase binding assays for your kinase of interest in order to avoid missing out on such compounds.
HTRF® kinase binding assays are easily set up, using a mix and measure detection format in the absence of ATP.
Direct displacement of the fluorescent-inhibitor from the ATP binding pocket is measured, enabling the affinity of your inhibitor to be determined by performing dose response curves.
Purified kinase preparation is less critical compared to enzymatic assays, since measurements can be performed on kinases which have little or no activity.
How it works
Kinase-GST discovery kit assay principle
The binding of the tracers is detected in a sandwich assay format using a specific Anti GST antibody labeled with Europium Cryptate (donor), which binds to GST-tagged Kinase, and a red fluorescent tracer labelled with d2 (acceptor). The HTRF ratio (665/620) will increase upon the addition of more of the tracer, and will saturate depending on the dissociation constant (Kd) of the tracer to the GST-tagged kinase. The Kinase-GST binding Discovery Kit helps determine which tracer might be best suited to setting up your binding assay . The tracer with the best assay properties (depending on the Kd and assay window generated) will be chosen to perform competitive binding assays.

 
Kinase-GST discovery kit assay protocol
Saturation binding experiments with the three tracers (i.e. Staurosporine-Red, Dasatinib-Red, and Sunitinib-Red) can be run on 96- or 384-well plates (20 µL final volume). First, a dilution series ranging between 0 and 1 µM of tracer in the Kinase Binding Buffer is prepared in a 96-well non-binding plate. Next, 5 µL of Kinase Binding Buffer are dispensed into the final 96- or 384-well plate. Then 5 µL of GST tagged-Kinase are added, followed by 5 µL of Anti-GST Eu-cryptate. Finally, 5 µL of the red tracer solution are added. The HTRF ratio is measured after 1 H of incubation. Application-Biochemical Enzymatic Assay
Brand-HTRF
Detection Modality-HTRF
Product Group-Kit
Shipping Conditions-Shipped in Dry Ice
Target Class-Kinases
Target Species-Human
Technology-TR-FRET
Therapeutic Area-Metabolism/Diabetes Neuroscience Oncology & Inflammation
Unit Size-1 unit