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 6HIS-tagged 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 6HIS-tagged 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 BINDING
  • FLUORESCENT INHIBITOR SELECTION
  • KD DETERMINATION

Kinase-6HIS discovery kit assay principle

The binding of the tracers is detected in a sandwich assay format using a specific Anti 6HIS antibody labeled with Europium Cryptate (donor), which binds to 6HIS-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 6HIS-tagged kinase.

The Kinase-6HIS 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.

Assay principle of kinase binding for 6HIS discovery kit

Kinase-6HIS discovery kit assay protocol

Saturation binding experiments on the three tracers (i.e. Staurosporine-Red, Dasatinib-Red, and Sunitinib-Red) can be run in 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 6HIS tagged-Kinase are added, followed by 5 µL of Anti-6HIS 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 6HIS discovery kit

Saturation Binding FGFR1-6HIS

​​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 22 nM on 5 nM FGFR1-6HIS.

Kd curves of staurosporine-Red on FGFR1-6His

Saturation Binding PDGFRb-6HIS

​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 45 nM on 5 nM PDGFRb-6HIS. 

Kd curves of Sunitinib-Red on PDGFRb-6His
The ultimate guide to successful kinase binding experiments

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Cisbio Product Catalog 2019

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