Kinase-biotin Binding Discovery Kit HTRF®

The binding of the tracers is detected in a sandwich assay format using a specific streptavidin labeled with Europium Cryptate (donor), which binds to N-terminal biotinylated 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 N-terminal biotinylated kinase .

The Kinase-biotin binding Discovery Kit helps determine which tracer might be best suited to setting up a 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.

Saturation binding experiments on 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 of tracer ranging between 0 and 1  µM 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 N-terminal biotinylated-Kinase are added, followed by 5 µL of Streptavidin Eu-cryptate. Finally, 5 µL of the red tracer solution are added. 

The HTRF ratio is measured after 1 H of incubation.

A typical saturation binding experiment is performed using final tracer concentrations between 0 and 250 nM, and measuring total- and non-specific binding signals. Subtracting the non-specific from the total binding signal gives the specific signal, which can be analysed to give the Kd. Here an example is shown where the best tracer for inhibitor studies proved to be Sunitinib-Red, with a Kd of 22 nM on 5 nM KIT-BTN. ​​

​A typical saturation binding experiment is performed using final tracer concentrations between 0 and 250 nM, and measuring total- and non-specific binding signals. Subtracting the non-specific from the total binding signal gives the specific signal, which can be analysed to give the Kd. Here an example is shown where the best tracer for inhibitor studies proved to be Staurosporine-Red, with a Kd of 35 nM on 5 nM SRC-BTN. ​​

​A typical saturation binding experiment is performed using final tracer concentrations between 0 and 250 nM, and measuring total- and non-specific binding signals. Subtracting the non-specific from the total binding signal gives the specific signal which can be analysed to give the Kd. Here an example is shown where the best tracer for inhibitor studies proved to be Dasatinib-Red, with a Kd of 52 nM on 5 nM BRAF-BTN. ​​