The Kinase Binding Buffer is a spare part intended for use in the HTRF® Kinase Binding platform only.

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The Kinase Binding Buffer is a spare part intended for use in the HTRF® Kinase Binding platform only.

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Overview

​The Kinase Binding Buffer is used to perform saturation binding (Kd-determination) and/or competitive binding assays within the HTRF® Kinase Binding platform only.

Benefits

  • PRODUCED BY MANUFACTURER
  • STRINGENT QUALITY CONTROL

Kd determination assay principal

The binding of a fluorescent tracer is detected in a sandwich assay format using either a specific Anti-GST, 6HIS antibody or Streptavidin labeled with Europium Cryptate (donor), which bind to the tagged Kinase, and a red fluorescent tracer labelled with d2 (acceptor). The detection principle is based on HTRF® technology.  The HTRF ratio (665/620) will increase upon the addition of more tracer, and will saturate depending on the dissociation constant (Kd ) of the tracer to the tagged kinase. When an inhibitor of the kinase is added, the tracer will be displaced and the HTRF signal will disappear, depending on the dose.

IC50/Ki determination assay protocol

​Pharmacological evaluation of inhibitors of interest can be run in 96- or 384-well plates.

First, a dilution series of inhibitor ranging between 40 µM and 0.23 nM is prepared, and 5 µL of each concentration are dispensed into the plate. Next, 5 µL of tagged-Kinase are added, followed by 5 µL of anti-tag Eu-cryptate. Finally, 5 µL of tracer solution are added, prepared at 4x the final concentration.  The HTRF ratio is measured after 1 H of incubation.

Analyses of the data give typical dose response curves ranging between 10 µM and 56 pM, enabling an  evaluation of the IC50/Ki values for the inhibitor of interest.

The ultimate guide to successful kinase binding experiments

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

3 app notes on kinase inhibitor selection for kinase binding assays

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

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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