The ability of hNT-3 to induce phosphorylation of TrkB was assessed. After starvation, TrkB-transfected NIH/3T3 cells were treated with increasing concentrations of hNT-3 for 5 minutes. Cells were lysed, and phospho-TrkB was quatified using PathScan® Phospho-TrkB (panTyr) Sandwhich ELISA Kit #7108. OD450-OD650 is shown.
The proliferation of TrkC-transfected NIH/3T3 cells treated with increasing concentrations of hNT-3 was assessed. After 24 hr treatment, cells were labeled with BrdU for 4 hrs. BrdU incorporation was determined by ELISA and the OD450-OD690 was determined.
The purity of recombinant hNT-3 was determined by SDS-PAGE of 6 µg reduced (+) and non-reduced (-) recombinant hNT-3 and staining overnight with Coomassie Blue.
Western blot analysis of extracts from TrkB-transfected NIH/3T3 cells untreated and treated with hNT-3 for 5 minutes, using Phospho-TrkA (Tyr674/675)/TrkB (Tyr706/707) (C50F3) Rabbit mAb #4621 (upper) or TrkB (80E3) Rabbit mAb #4603 (lower).
With carrier: Lyophilized from a 0.22 μm filtered solution of 20 mM phosphate, pH 8.0 containing 500 mM NaCl and 20 μg BSA per 1 μg hNT-3. Carrier free: Lyophilized from a 0.22 μm filtered solution of 20 mM phosphate, pH 8.0 containing 500 mM NaCl.
Stable in lyophilized state at -20°C for 1 year after receipt. Sterile stock solutions reconstituted with carrier protein are stable at 4°C for 2 months and at -20°C for 6 months. Avoid repeated freeze-thaw cycles.Maintain sterility. Storage at -20°C should be in a manual defrost freezer.
>98% as determined by SDS-PAGE of 6 μg reduced (+) and non-reduced (-) recombinant hNT-3. All lots are greater than 98% pure.
Less than 0.01 ng endotoxin/1 μg hNT-3.
The bioactivity of recombinant hNT-3 was determined in a TrkC-transfected NIH/3T3 cell proliferation assay. The ED50 of each lot is between 0.5 - 5 ng/ml.
Recombinant hNT-3 does not have a Met on the amino terminus and has a calculated MW of 13,625. DTT-reduced and non-reduced protein migrate as 14 kDa polypeptides. The expected amino-terminal YAEHK of recombinant hNT-3 was verified by amino acid sequencing.
Recombinant human Neurotrophin-3 (hNT-3) Tyr139-Thr257 (Accession #NP_002518) was produced in E.coli at Cell Signaling Technology.
NT-3 is a member of the structurally related neurotrophin family of proteins, which includes β-NGF, BDNF and NT-4 (1). NT-3 is expressed in a number of cell types including neuronal cells, eosinophils, and melanocytes (1-3). NT-3 is required for the development of peripheral sensory neurons (4). NT-3 is secreted from cells as a precursor protein, which is proteolytically cleaved into the mature form (1). NT-3 signaling is mediated through two distinct receptors, the neurotrophin receptor p75NTR and the Trk tyrosine kinase receptor TrkC. While all neurotrophins bind to the p75NTR receptor, NT-3 preferentially binds to TrkC and, with lesser affinity, TrkB (1).
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