Technology - Cell-based Kinases
PathHunter® Cell-based Kinase Assays are functional, whole cell assays designed for compound screening and profiling against high value kinase targets in the areas of oncology, neurological disorders, metabolic disease and inflammation. These fully optimized, target-validated assays allow you to study kinase activation, receptor internalization or screen for novel kinase inhibitors using a simple and quantitative EFC-based chemiluminescent assay.
Assay Principles
PathHunter® Receptor Tyrosine Kinase Assay Principle
These cell lines express a full length receptor tyrosine kinase (RTK) protein fused in frame with a small epitope tag called ProLink™ on the intracellular C-terminus of the receptor. Cells co-express a fusion protein of the SH2 domain protein and the larger, N-terminal deletion mutant of β-gal (called enzyme acceptor or EA).
Activation of receptor results in dimerization and phosphorylation of the RTK and subsequent interaction with the SH2 protein which forces complementation of the two enzyme fragments, resulting in the formation of an active β-gal enzyme. This action leads to an increase in enzyme activity that can be measured using chemiluminescent PathHunter® Detection Reagents.
Schematic of PathHunter® Receptor Tyrosine Kinase Assay
Use of full length receptor protein allows identification of anti-ligand/anti-receptor neutralizing and anti-dimerization antibodies [click graph to enlarge].
It is also ideal for small molecule inhibitors [click graph to enlarge].
PathHunter® Cytosolic Tyrosine Kinase Assay Principle
Unlike receptor tyrosine kinases, cytokine receptors lack kinase activity and are phosphorylated by cytosolic tyrosine kinases such as the JAK kinases. To detect the phosphorylation state of cytokine receptors, PathHunter® Cytosolic Tyrosine Kinase cell lines are engineered to express a full length cytokine receptor fused in frame on the C-terminus with a small epitope tag of b-gal called ProLink™. Cells co-express a phosphotyrosine binding domain fused to the complementing fragment, EA.
Upon activation and phosphorylation of the receptor by the cytosolic tyrosine kinase, the SH2 domain binds to the phosphorylated residues and complementation of the two enzyme fragments results in the formation of an active β-gal enzyme leading to an increase in enzyme activity that can be measured using chemiluminescent PathHunter® Detection Reagents. Specificity for a kinase can be obtained by co-expression of specific tyrosine kinases.
Schematic of PathHunter® Cytosolic Tyrosine Kinase Assay
The PathHunter Cell-based Kinase Advantage
Related Products - PathHunter RTK & CTK Cell Lines & Assays
Clonal Cell Lines
Unique, flexible whole cell assay platform for measuring kinase activation or inhibition for kinases screening and profiling.
Assay-Ready (Ready-to-Use) PathHunter eXpress® Kits
Simple mix-and-read microtiter plate-based functional whole-cell tyrosine kinase assays.