Complex of recombinant human proteins TPM3 (tropomyosin 3) encompassing amino acids 1-258, and TRKA (neurotrophic receptor tyrosine kinase 1) encompassing amino acids 399-end. This construct contains an N-terminal GST tag. This recombinant protein was affinity purified and is kinase active.
50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM glutathione, 0.1 mM EDTA, 0.25 mM DTT, 0.1 mM PMSF, 25% glycerol.
MW
105 kDa
Amino Acids
TPM3: 1-258, TRKA: 399-end
Specific Activity
13 pmol/min/µg
Genbank #
TPM3: NM_152263, TRKA: NM_002529
UniProt #
TPM3: P06753; TRKA: P04629
Tag(s)
N-Terminal GST-Tag
Background
TRKA is a member of the neurotrophic tyrosine kinase receptor (NTKR) family which is a membrane-bound receptor that, upon neurotrophin binding, phosphorylates itself and members of the MAPK pathway. TRKA has a crucial role in the development and function of the nociceptive reception system as well as establishment of thermoregulation via sweating in humans. The oncogenic rearrangement of 5′TPM3-3′NTRK1 fusion gene has been found in papillary thyroid carcinomas.
References
1. Smeyne, R. J. et.al: Severe sensory and sympathetic neuropathies in mice carrying a disrupted Trk/NGF receptor gene. Nature 368: 246-249, 1994.
2. Marta G. B. et.al: A Sequence Analysis of the Genomic Regions Involved in the Rearrangements between TPM3 and NTRK1 Genes Producing TRK Oncogenes in Papillary Thyroid Carcinomas. Genomics, Volume 28, Issue 1, 1 July 1995, Pages 15–24
Storage/Stability
At least 6 months at -80°C.
Assay Conditions
Kinase activity was measured using a Poly (Glu4, Tyr1) synthetic peptide substrate diluted in distilled water to a final concentration of 1 mg/ml. Increasing amounts of kinase were mixed with Poly (Glu4,Tyr1) synthetic peptide substrate with a final concentration of 200 µg/ml in a buffer containing 5 mM MOPS, pH 7.2, 2.5 mM β-glycerol-phosphate, 5 mM MgCl2, 1 mM EGTA, 0.4 mM EDTA and 0.05 mM fresh DTT to a final volume of 20 uL. The reaction was initiated by addition of 5 μl of [33P]-ATP diluted in kinase buffer: 6 ml kinase buffer containing 1 mCi [33P]-ATP, 0.25 mM ATP, 25 mM MOPS, pH 7.2, 12.5 mM β-glycero-phosphate, 25 mM MgCl2, 5 mM EGTA, 2 mM EDTA, and 0.25 mM fresh DTT. After incubating for 30°C for 15 minutes, the reaction was terminated by spotting 20 μL of the mixture onto phosphocellulose paper strips that were fixed in 1% phosphoric acid and washed three times. Radioactivity was determined using a scintillation counter. The blank was determined from a “no substrate” sample.
Instructions for Use
Thaw on ice and gently mix prior to use. DO NOT VORTEX. Perform a quick spin before opening. Aliquot into small volumes and flash freeze for long term storage. Avoid multiple freeze/thaw cycles.
Applications
Useful for the study of enzyme kinetics, screening inhibitors, and selectivity profiling.
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