EphA2 Kinase Inhibitor Screening Assay Kit

Intended Use

The EphA2 Kinase Inhibitor Screening Assay Kit is designed to measure the activities of recombinant catalytic domain of EphA2 for the rapid and sensitive evaluation of inhibitors or activators. The phosphotyrosine specific monoclonal antibody used in this assay kit has been demonstrated to recognize the phosphotyrosine residue in recombinant "Tyrosine kinase-substrate-1", which is efficiently phosphorylated by EphA2 in vitro.

Applications of this kit include:
1) Screening inhibitors or activators of recombinant catalytic domain of EphA2 Kinase.
2) Detecting the effects of pharmacological agents on recombinant catalytic domain of EphA2 Kinase.

This assay kit is for research use only and not for use in diagnostic or therapeutic procedures.

Storage

Upon receipt store all components at 4°C.
Don't expose reagents to excessive light.

Performance Characteristics

The EphA2 Kinase Inhibitor Screening Assay Kit has been shown to detect the kinase activity of recombinant catalytic domain of EphA2. The assay shows good linearity of sample response.

General Description

The EphA2 receptor tyrosine kinase belongs to the Eph receptor family. The Eph family contains at least 14 members that are type I transmembrane glycoproteins. Upon cell-cell contact, Eph receptors interact with their ligands called ephrins. Glycosylphosphatidyl inositol-linked ephrin-A ligands bind to EphA receptors and transmembrane ephrin-B ligands bind to EphB receptors. Eph-ephrin signaling mediates contact-dependent cell interactions involved in the spatial organization of neurons and neural crest cells, embryogenesis, tissue patterning, and blood vessels formation.
EphA2 is overexpressed in several human cancers such as breast, colon, lung, prostate, gastric carcinoma, and metastatic melanoma but not in nonmalignant breast, prostate, and skin lesions. EphA2 overexpression correlates with malignant transformation, poor prognosis, metastatic progression, and p53-mediated apoptosis. EphA2 has been detected in tumor neovasculature and was demonstrated to play a role in tumor angiogenesis. The large number of human cancers expressing EphA2 and the association with tumor vascularization and metastatization prompt EphA2 as an appealing target for tumor immunotherapy.
EphA2 is also functionally altered in malignant cells. The EphA2 in malignant cells is generally not tyrosine phosphorylated. This EphA2 retains enzymatic activity, but the tyrosine-unphosphorylated EphA2 promotes tumor cell growth, invasion, and survival, while tyrosine-phosphorylated EphA2 negatively regulates cell growth and invasion.

Measurement of EphA2 Kinase activity

The protocol generally regarded as most sensitive for the quantitative measurement of EphA2 kinase activity involves incubation of the EphA2 kinase sample with substrate, either a natural or synthetic polypeptide (such as poly[Glu,Tyr]4:1), in the presence of Mg2+, Mn2+ and 32P-labeled ATP. The reaction is terminated by "spotting" a sample onto a filter paper disc, followed by immersion in acid to precipitate the radiolabeled product. The filter papers are then washed extensively to remove unincorporated radiolabel and the radioactivity is counted. While sensitive, this method is labor-intensive, generates hazardous radioactive waste, and depends on a radioisotope of short half-life. It is particularly unsuitable when kinase assays are only performed on an infrequent basis. The Research Product EphA2 Kinase Inhibitor Screening Assay Kit uses a horseradish peroxidase coupled anti-phosphotyrosine monoclonal antibody as a reporter molecule in a 96-wells ELISA format. This assay provides a non-isotopic, sensitive and specific method to detect kinase activity of recombinant EphA2 catalytic domain.