Revvity, 62TK0PEB, HTRF KinEASE TK Kit, 1,000 Assay Points

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 TK assay has been validated on 71 Tyrosine kinases, using a single generic substrate. The assay validation section of this page lists them and provides technical tips for setup. 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. The TK generic substrate is made of a sequence of repetitive motifs recognized by most tyrosine kinases, and by the specific conjugated antibody once phosphorylated. 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.