Technologies & Platforms / Enzyme Fragment Complementation Technology / PathHunter EFC Cell-Based Assay Platform / Protein:Protein Interactions / Kinases RTK & CTK

PathHunter® Kinase Cell Based Assay Platform

PathHunter receptor tyrosine and cytosolic tyrosine kinase assays are whole cell high-throughput assays that utilize the enzyme fragment complementation technology to look at kinase activation. Upon stimulation, the receptor is activated resulting in phosphorylation and binding by an SH2-EA fusion protein.  This forces complementation of the small peptide tag, called Prolink™, and the larger enzyme acceptor (EA) fragment to form a functional β-gal enzyme, which hydrolyzes substrate to generate a chemiluminescent signal.

Technology Principle

Receptor Tyrosine Kinase

Cytokine Receptor Kinase
rkp assay principle ctk assay principle
In this PathHunter® assay approach, a small peptide epitope (ProLink™) is expressed recombinantly on the intracellular C-terminus of the Receptor Tyrosine Kinase. One of the many different partner proteins containing SH2 domains is co-expressed with a larger sequence, termed enzyme acceptor (EA). Ligand induced activation of the receptor causes either homo- or hetero-dimerization of the receptor resulting in crossphosphorylation. The SH2-EA fusion protein then binds the phosphorylated receptor forcing complementation of the PK and EA fragment. This interaction generates an active β-galactosidase enzyme, which is detected using a chemiluminescent substrate.


Performance Data for PathHunter® RTK & CTK Kinase Assays

Insulin Receptor

      

JAK Analysis
insulin receptor  
The insulin receptor is constitutively dimerized but undergoes a conformational change to become active in the presence of insulin and this can be inhibited by the small molecule antagonists staurosporine and HNMPA [click graph to enlarge].
  
The CSF3R cell line was preincubated with antagonists such as staurosporine, lestaurtinib and pyridone 6, P6, DBI (Jak inhibitor 1) and then challenged with EC80 of agonist G-CSF. A dose dependent inhibition was observed with antagonist indicating that the assay can be used effectively to profile or screen inhibitors against the JAK molecule [click graph to enlarge].

Features & Benefits

  • Single functional readout broadly applicable to different kinase receptors and groups
  • Stable cell lines and a one-step, no wash procedure increase reproducibility
  • Ideal for large-molecule therapeutics and small-molecule inhibitors
  • High specificity, as tagged receptor eliminates background from endogenous receptors
  • High-quality reproducible data, due to superior assay windows and robust performance
  • Ability to identify various inhibitors including allosteric, ligand- and ATP-competitive