Description
The HTRF KinEASE kit series provides a simple biochemical approach to study kinase activity, screen for inhibitors and characterize them. Three STK and one TK kits, offer a semi-universal method for assessing phosphorylation on Serine/Threonine and Tyrosine residues, respectively. The HTRF KinEASE STK S2 assay has been validated on 18 Serine/Threonine kinases. The assay validation section of this page lists them and provides technical tips for setup. The kit series also include a Discovery kit to help determine which of three STK kits is appropriate when working with a non-validated enzyme.
HTRF assays offer many advantages over other technologies:
Homogeneous add-and-read format
No wash steps
Low background
Straightforward miniaturization from 96- or 384-well microplates to high density assay formats such as 384-well low volume and 1536-well plates
Stable signal, providing flexibility in time of readout or size of assays
Assay principle
Each HTRF KinEASE kit is based on the same assay format, and uses a universal biotinylated substrate, a phospho-specific Eu-cryptate-labeled monoclonal antibody, and SA-XL665. All three STK peptidic substrates are made of the same sequence for antibody detection, next to a variable sequence recognized by enzymes. The targeted residue for phosphorylation lies in between as part of both sequences for enzymatic activity (unphosphorylated) and antibody detection (phosphorylated). It has been shown that 3 different variable sequences could enable the study of most Serine/Threonine kinases.
Assay protocol
As shown here, all HTRF KinEASE assays work the same way and involve two steps:
Step 1: Kinase reaction The kinase is incubated in the presence or absence of compounds and the appropriate substrate. The supplemented enzymatic buffer is added in the kinase buffer for tyr kinases only. ATP is added to start the reaction.
Step 2: Detection Eu-cryptate and XL665 conjugates are added. The detection buffer contains EDTA to stop the enzymatic reaction.
