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

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  • Developed and tested to Cisbio's high quality standards Developed and tested to Cisbio's high quality standards

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.

Principal of kinase binding saturation assay using staurosporine-red

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.

Protocol for kinase binding inhibitor assay using staurosporine-red
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