HTRF Kinase Binding Buffer, 20 ml

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.