PathHunter® Receptor Tyrosine Kinase Assay Internalization Platform

Receptor Tyrosine Kinases are implicated in many disease states such as cancer, metabolic disease, inflammatory disease, cardiovascular disease etc. DiscoveRx has utilized its proprietary enzyme fragment complementation (EFC) technology to develop cell-based functional assays to monitor RTK internalization. This assay platform does not require antibodies or washes and can be easily adapted to a one-step HTS-friendly assay.

Technology Principle

PathHunter Receptor Tyrosine Kinase Assay Platform
The small enzyme fragment of β-gal called ProLink™ (PK) is appended to the C-terminus of the receptor tyrosine kinase. The larger, complementing enzyme fragment termed Enzyme Acceptor (EA) is localized exclusively on the surface of cellular endosomes. Stimulation of the RTK results in receptor activation and subsequent internalization and trafficking to cellular endosomes. These events drive complementation of the two of β-gal enzyme fragments. The resultant increase of β-gal enzyme activity can be easily measured using chemiluminescent PathHunter® Detection Reagents.

Assay Performance & Metrics

EGFR HEK Endo-PK

INSR U20S Endo-EA

Dose curves demonstrating the activation and consequent internalization of insulin receptor (INSR) and epidermal growth factor receptor (EGFR) as a response to respective agonist stimulation.

Features & Benefits

  • Ligand induced functional assay
  • Whole cell, HTS-friendly, chemiluminescent assay
  • Uses full length receptor tyrosine kinase
  • No wash, single addition