PathHunter receptor tyrosine and cytosolic tyrosine kinase assays are whole cell, high-throughput assays that utilize the enzyme fragment complementation technology based on a split β-galactosidase (β-gal) enzyme system to look at kinase activation.
In this PathHunter assay approach, a small inactive peptide fragment of the β-galactosidase enzyme called ProLink™ (PK) is expressed recombinantly on the intracellular C-terminus of the Receptor Tyrosine Kinase (left image) or cytokine receptor (right image). One of the many different partner proteins containing SH2 domains is co-expressed with a larger fragment of β-gal, termed enzyme acceptor (EA). Ligand induced activation of the receptor causes either homo- or hetero-dimerization of the receptor resulting in cross-phosphorylation. 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.
DiscoverX offers a variety of products and services to study kinases.
Kinase Cell Lines
Kinase Assay Ready Kits
- RTKs and CTKs activity and functional assays
Related Products and Services
Advantages of Kinase Cell-Based Assays
- Broadly Applicable - Identify various ligands including anti-receptor, anti-ligand, or activating antibodies; non-ATP pocket binders (allosteric modulators); ligand binding inhibitors (ATP-competitors); or dimerization inhibitors
- Accurate Reproducibility - Superior quality, reproducible data with large assay windows and robust performance
- High Specificity - Tagged, full-length tyrosine kinase eliminates background from endogenous tyrosine kinases
Create Your Own SH2-Protein Recruitment Cell-Based Assays Using Engineered Parental Cell Lines
Generate a PathHunter SH2-protein recruitment assay by first creating a plasmid vector with your target protein of choice (e.g. RTK in this example) tagged with the enzyme donor ProLink™ (PK). Simply transfect this plasmid into a PathHunter SH2-recuitment EA parental cell line containing an EA-SH2 protein, and then perform a kinase activity assay in the presence of a ligand.
Evaluate Multiple Mechanisms of Receptor and Cytosolic Tyrosine Kinase Activation Using Stable Cell Lines
A. The insulin receptor is constitutively dimerized, but undergoes a conformational change to become active in the presence of insulin (agonist) and this can be inhibited by the small molecule antagonists staurosporine and HNMPA. B. The colony stimulating factor 3 receptor (CSF3R-JAK1) cell line was preincubated with antagonists staurosporine, lestaurtinib and pyridone 6, P6, DBI (a 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 cytosolic tyrosine kinase JAK1.
Easy-to-Use – Simple, One-Step Add and Read Protocol
Simple no wash, add, mix, and read protocols with a chemiluminescent output that can be read on any benchtop luminometer. Using the kinase cell-based assays, you can eliminate the need to load target cells prior to every experiment, eliminate the use of radioactivity, and reduce the number of workflow steps, increasing the efficiency of the lab.