TrkA Kinase Inhibitor Screening Assay Kit

Intended Use

The TrkA Kinase Inhibitor Screening Assay Kit is designed to measure the activities of recombinant catalytic domain of TrkA 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 TrkA in vitro.

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

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

General Description

The Trk (tropomyosin receptor kinase) receptors belong to the family of receptor tyrosine kinases, and three trk genes have been identified in mammals. The TrkA protooncogene was first identified as an nerve growth factor receptor (NGFR), followed by TrkB and TrkC. Nerve growth factor (NGF) is the preferred ligand for TrkA, brain-derived neurotrophic factor (BDNF) and Neurotrophin-4/5 (NT-4/5) are preferred for TrkB, and Neurotrophin-3 (NT-3) for TrkC. These specificities are not absolute, and NT-3 is also a ligand for TrkA and TrkB. These Trk receptors are transmembrane glycoproteins of ~140 kD. They are tyrosine kinases with an extracellular ligand-binding domain containing multiple repeats of leucine-rich motifs, two cysteine clusters (C1, C2), two immunoglobulin-like domains (Ig1, Ig2), and a single transmembrane domain. The tyrosine kinase domains are highly related (~80% amino acid identity), however the extracellular domains are more divergent (~30%).
Signaling through the Trk receptors-the main pathways. Neurotrophin binding to Trk receptors triggers dimerization leading to the activation of different signaling pathways through recruitment of various adapter molecules. The main binding sites for Trk substrates are two tyrosine residues that, on activation of the Trk receptors, become phosphorylated. Two complexes of adapter molecules bind to the tyrosine residue located in the juxtamembrane region of the Trk receptor, the Shc/Grb2/SOS and the FRS2/SHP-2/Grb2/SOS complex. Ras/Raf/MEK/MAPK induces the differentiation of neurons and neurite growth.

Measurement of TrkA Kinase activity

The protocol generally regarded as most sensitive for the quantitative measurement of TrkA kinase activity involves incubation of the TrkA 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 TrkA 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 TrkA catalytic domain.