Human Total TrkA DuoSet IC ELISA
Human Total TrkA DuoSet IC ELISA Summary
* Provided that the recommended microplates, buffers, diluents, substrates and solutions are used, and the assay is run as summarized in the Assay Procedure provided.
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- Optimized capture and detection antibody pairings with recommended concentrations save lengthy development time
- Development protocols are provided to guide further assay optimization
- Assay can be customized to your specific needs
- Available in 2, 5, and 15- (96-well) plate pack sizes
- Economical alternative to Western blot
- Capture Antibody
- Conjugated Detection Antibody
- Calibrated Immunoassay Standard or Control
Other Reagents Required
PBS: (Catalog # DY006), or 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2O4, pH 7.2 - 7.4, 0.2 µm filtered
Wash Buffer: (Catalog # WA126), or equivalent
Substrate Solution: 1:1 mixture of Color Reagent A (H2O2) and Color Reagent B (Tetramethylbenzidine) (Catalog # DY999)
Stop Solution: 2 N H2SO4 (Catalog # DY994)
Microplates: From Costar EIA Plate (Costar Catalog # 2592) or R&D Systems (Catalog # DY990), or equivalent
Plate Sealers: ELISA Plate Sealers (Catalog # DY992), or equivalent
*For the Lysis Buffer, IC Diluent, and Blocking BUffer recommended for a specific DuoSet ELISA Development Kit, please see the product
The Human Total TrkA DuoSet® IC ELISA is more sensitive than immunoprecipitation (IP)-Western blot analysis. Lysates prepared from C6 rat glioma cells transfected with human TrkA (C6-hTrkA) were serially diluted and analyzed by (A) IP-Western blot and (B) this DuoSet® IC ELISA. IPs were performed using an anti-TrkA monoclonal antibody and goat anti-mouse agarose. Immunoblots were incubated with a biotinylated anti-TrkA polyclonal antibody to detect total TrkA. Bands were visualized with Streptavidin-HRP (Catalog # DY998) followed by chemiluminescent detection. Human TrkA can be detected by this Human Total TrkA DuoSet® IC ELISA by using approximately 2 to 4 times less lysate than is needed for a conventional IP-Western Blot.
Preparation and Storage
TrkA, the product of the protooncogene trk, is a member of the neurotrophic tyrosine kinase receptor family that has three members. TrkA, TrkB and TrkC preferentially bind NGF, NT-4 and BDNF, and NT-3, respectively. All Trk family proteins share a conserved complex subdomain organization consisting of a signal peptide, two cysteine-rich domains, a cluster of three leucine-rich motifs, and two immunoglobulin-like domains in the extracellular region, as well as an intracellular region that contains the tyrosine kinase domain.
Two distinct TrkA isoforms that differ by virtue of a 6 amino acid insertion in their extracellular domain have been identified. The longer TrkA isoform is the only isoform expressed within neuronal tissues whereas the shorter TrkA is expressed mainly in non neuronal tissues. NGF binds to TrkA with low affinity and activates its cytoplasmic kinase, initiating a signaling cascade that mediates neuronal survival and differentiation. Higher affinity binding of NGF requires the coexpression of TrkA with the p75 NGF receptor (NGFR), a member of the tumor necrosis factor receptor superfamily. NGFR binds all neurotrophins with low affinity and modulates Trk activity as well as alters the specificity of Trk receptors for their ligands. NGFR can also mediate cell death when expressed independent of Trk.
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