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 GST-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 GST-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.

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.

Benefits

  • EQUILIBRIUM BINDING
  • FLUORESCENT INHIBITOR SELECTION
  • KD DETERMINATION

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.

Assay principle of kinase binding for GST discovery kit

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.

Assay protocol of kinase binding for GST discovery kit

Saturation Binding SRC-GST

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 43 nM on 5 nM SRC-GST. ​​

Kd curves of Staurosporine-Red on SRC-GST

Saturation Binding PDGFRb-GST

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 56 nM on 5 nM PDGFRb-GST. ​​

Inhibitor effect of various inhibitors on Sunitinib-red /PDGFRb-GST binding

Saturation Binding BRAF-GST

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 22 nM on 5 nM BRAF-GST.

Kd curves of Dasatinib-Red on BRAF-GST
3 app notes on kinase inhibitor selection for kinase binding assays

Determine the best tracer to your kinase binding assay - Application Notes

The ultimate guide to successful kinase binding experiments

Tips for getting the best performance from your Kinase binding assay - Guides

Cisbio Product Catalog 2019

All your HTRF assays in one document! - Catalog

A guide to Homogeneous Time Resolved Fluorescence

General principles of HTRF - Guides

The essential white paper to expand your knowledge on kinases

Learn about cutting edge kinase research in this free white paper - Guides

How HTRF compares to Western Blot and ELISA

Get the brochure about technology comparison. - Brochures

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