Kd determination assay principal

The binding of Sunitinib-Red is detected in a sandwich assay format using the Anti Tag labeled with Europium Cryptate (donor), which binds 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 of the tracer, and will saturate depending on the dissociation constant (Kd) of the tracer to the tagged kinase.

​​​To know whether Sunitinib-Red is the tracer best suited to your tagged kinase of interest, we advise you to try the Kinase Binding Discovery kits.

Kd determination assay protocol

Saturation binding experiments of Sunitinib-Red can be run in 96- or 384-well plates (20 µL final volume).

First, a dilution series ranging between 0 and 1 µM of Sunitinib-Red 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 tagged-Kinase are added, followed by 5 µL of Anti-tag Eu-cryptate. Finally, 5 µL of the red tracer solution are added.

The HTRF ratio is measured after 1H of incubation.

IC50/Ki determination assay principle

The binding of Sunitinib-Red is detected in a sandwich assay format using a specific Anti-GST, 6HIS antibody or Streptavidin labeled with Europium Cryptate (donor), which binds to the tagged Kinase, and a red fluorescent Sunitinib labelled with d2 (acceptor).

The detection principle is based on HTRF® technology. The HTRF ratio (665/620) will increase upon the addition of more Sunitinib-Red, and will saturate depending on the dissociation constant (Kd ) of Sunitinib-Red to the tagged kinase.

When an inhibitor of the kinase is added, Sunitinib-Red 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 Sunitinib-Red 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.