MAb Anti 6HIS-Eu cryptate Kinase Binding HTRF®

​The binding of the tracers is detected in a sandwich assay format using the Anti 6HIS antibody labeled with Europium Cryptate (donor), which binds to the 6HIS-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 6HIS-tagged kinase. 

Saturation binding experiments on the three tracers (i.e. Staurosporine-Red, Dasatinib-Red, and Sunitinib-Red) can be run in 96- or 384-well plates (20 µL final volume).

First, a dilution series of tracer ranging between 0 and 1  µM 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 6HIS tagged-Kinase are added, followed by 5 µL of Anti-6HIS Eu-cryptate. Finally, 5 µL of the red tracer solution are added. 

The HTRF ratio is measured after 1 H of incubation.

Principle of HTRF® Kinase binding assay (IC50/Ki-determination). 

The binding of the tracers is detected in a sandwich assay format using the Anti-6HIS 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. When an inhibitor of the kinase is added, the tracer will be displaced and the HTRF signal will disappear, depending on the dose.

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

First a dilution series ranging between 40 µM and 0.23 nM of inhibitor  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-6HIS Eu-cryptate. Finally, 5 µL of tracer solution are added, prepared at 4x the final concentration.  The HTRF ratio is measured after 1H 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.

​​​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 22 nM on 5 nM FGFR1-6HIS.

D​ose response curves of various known kinase inhibitors (Staurosporine, Dasatinib,  PP2, Imatinib, Tozasertib, Sunitinib, Gefitinib, and Sorafenib) were measured using Sunitinib-Red at its Kd (45 nM) on 5 nM PDGFRb-6HIS.  Staurosporine, Dasatinib, Sunitinib, and Imatinib show showed high potencies, in good correlation with literature values.