This kit is intended for the quantitative measurement of the dissociation constant (Kd) of three different tracers (Staurosporine-Red, Dasatinib-Red and/or Sunitinib-Red) on N-terminal biotinylated kinases, using HTRF® technology.

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  • No-wash No-wash
  • Ease-of-use Ease-of-use
  • Under 2h bench time Under 2h bench time

This kit is intended for the quantitative measurement of the dissociation constant (Kd) of three different tracers (Staurosporine-Red, Dasatinib-Red and/or Sunitinib-Red) on N-terminal biotinylated kinases, using HTRF® technology.

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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.

Moreover the 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 determination of the affinity of your inhibitor 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.

Benefits

  • EQUILIBRIUM AND KINETIC BINDING
  • FLUORESCENT INHIBITOR SELECTION
  • KD DETERMINATION

Kinase-biotin discovery kit assay principle

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.

Kinase-biotin discovery kit assay protocol

Assay principle of kinase binding for biotin discovery kit

Kinase-biotin discovery kit assay protocol

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.

Assay protocol of kinase binding for biotin discovery kit

Saturation Binding KIT-Biotin

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. ​​

Kd curves of Sunitinib-Red on KIT-Biotin

Saturation Binding SRC-Biotin

​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. ​​

Kd curves of Staurosporine-Red on SRC-Biotin

Saturation Binding BRAF-Biotin

​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. ​​

Kd curves of Dasatinib-Red on BRAF-Biotin
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