Assay Type
Solid Phase Sandwich ELISA
Format
96-well strip plate
Assay Length
4 hours 40 mins (after plate preparation)
Sample Type & Volume Required
Cell lysates (100 µL)
Range
156.00 - 10,000 pg/mL
Sufficient Materials
Kits available for two, five, or fifteen 96-well plates*
Specificity
Please see the
* Provided that the recommended microplates, buffers, diluents, substrates and solutions are used, and the assay is run as summarized in the Assay Procedure provided.
Product Features
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
Kit Content
Capture Antibody
Conjugated Detection Antibody
Calibrated Immunoassay Standard or Control
Streptavidin-HRP
Other Reagents Required
PBS: (Catalog # ), or 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na 2HPO 4, 1.5 mM KH 2O 4, pH 7.2 - 7.4, 0.2 µm filtered
Wash Buffer: (Catalog # ), or equivalent
Lysis Buffer*
IC Diluent*
Blocking Buffer*
Substrate Solution: 1:1 mixture of Color Reagent A (H 2O 2) and Color Reagent B (Tetramethylbenzidine) (Catalog # )
Stop Solution: 2 N H 2SO 4 (Catalog # )
Microplates: From Costar EIA Plate (Costar Catalog # 2592) or R&D Systems (Catalog # ), or equivalent
Plate Sealers: ELISA Plate Sealers (Catalog # ), or equivalent
*For the Lysis Buffer, IC Diluent, and Blocking BUffer recommended for a specific DuoSet ELISA Development Kit, please see the product
Data Examples
Figure 1: 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 # ) 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
Storage
Store the unopened product at 2 - 8 °C. Do not use past expiration date.
Background: TrkA
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.
Entrez Gene IDs:
4914 (Human); 59109 (Rat);
Long Name:
Neurotrophic Tyrosine Kinase Receptor A
Aliases:
DKFZp781I14186; EC 2.7.10; EC 2.7.10.1; MTChigh affinity nerve growth factor receptor; Neurotrophic tyrosine kinase receptor type 1; neurotrophic tyrosine kinase, receptor, type 1; NTRK1; NTRK-1; p140-TrkA; TRK1-transforming tyrosine kinase protein; TrkA; Trk-A; TRKAOncogene TRK; TRKTRK1; tyrosine kinase receptor A
