Mouse Ntrk1 ELISA Kit (MOEB2415)- High Sensitivity

Product Name:	Mouse High affinity nerve growth factor receptor (Ntrk1) ELISA Kit
SKU:	MOEB2415
Size:	96T
Target:	Mouse High affinity nerve growth factor receptor (Ntrk1)
Synonyms:	Neurotrophic tyrosine kinase receptor type 1
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Mouse
Detection Range:	78-5000pg/mL
Sensitivity:	40.3pg/mL

Intra CV:

6.8%

Inter CV:

8.7%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	102-112%	100-110%	85-97%	105-115%
EDTA Plasma(N=5)	94-103%	100-113%	107-116%	96-108%
Heparin Plasma(N=5)	106-118%	110-120%	106-116%	107-119%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	86	80-92
Plasma	88	82-94

Function:

Receptor tyrosine kinase involved in the development and the maturation of the central and peripheral nervous systems through regulation of proliferation, differentiation and survival of sympathetic and nervous neurons. High affinity receptor for NGF which is its primary ligand, it can also bind and be activated by NTF3/neurotrophin-3. However, NTF3 only supports axonal extension through NTRK1 but has no effect on neuron survival. Upon dimeric NGF ligand-binding, undergoes homodimerization, autophosphorylation and activation. Recruits, phosphorylates and/or activates several downstream effectors including SHC1, FRS2, SH2B1, SH2B2 and PLCG1 that regulate distinct overlapping signaling cascades driving cell survival and differentiation. Through SHC1 and FRS2 activates a GRB2-Ras-MAPK cascade that regulates cell differentiation and survival. Through PLCG1 controls NF-Kappa-B activation and the transcription of genes involved in cell survival. Through SHC1 and SH2B1 controls a Ras-PI3 kinase-AKT1 signaling cascade that is also regulating survival. In absence of ligand and activation, may promote cell death, making the survival of neurons dependent on trophic factors.

Uniprot:	Q3UFB7
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant mouse High affinity nerve growth factor receptor
Sub Unit:	Exists in a dynamic equilibrium between monomeric (low affinity) and dimeric (high affinity) structures. Homodimerization is induced by binding of a NGF dimer (By similarity). Found in a complex, at least composed of KIDINS220, MAGI2, NTRK1 and RAPGEF2; the complex is mainly formed at late endosomes in a nerve growth factor (NGF)-dependent manner. Interacts with RAPGEF2; the interaction is strengthened after NGF stimulation. Interacts with SQSTM1; bridges NTRK1 to NGFR. Forms a ternary complex with NGFR and KIDINS220; this complex is affected by the expression levels of KIDINS220 and an increase in KIDINS220 expression leads to a decreased association of NGFR and NTRK1. Interacts (phosphorylated upon activation by NGF) with SHC1; mediates SHC1 phosphorylation and activation. Interacts (phosphorylated upon activation by NGF) with PLCG1; mediates PLCG1 phosphorylation and activation. Interacts (phosphorylated) with SH2B1 and SH2B2. Interacts with GRB2. Interacts with PIK3R1. Interacts with FRS2. Interacts with SORT1; may regulate NTRK1 anterograde axonal transport (By similarity). Interacts with SH2D1A; regulates NTRK1 (PubMed:16223723). Interacts with NRADD. Interacts with RAB7A. Interacts with PTPRS.
Subcellular Location:	Cell membrane Single-pass type I membrane protein Early endosome membrane Single-pass type I membrane protein Late endosome membrane Single-pass type I membrane protein Internalized to endosomes upon binding of NGF or NTF3 and further transported to the cell body via a retrograde axonal transport. Localized at cell membrane and early endosomes before nerve growth factor (NGF) stimulation. Recruited to late endosomes after NGF stimulation. Colocalized with RAPGEF2 at late endosomes.
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	TrkA: a receptor tyrosine kinase of the Trk family. High affinity receptor for nerve growth factor, neurotrophin-3 and neurotrophin-4/5 but not brain- derived neurotrophic factor. Known substrates include Shc, PI-3K, and PLC-gamma-1. Has a crucial role in the development and function of the nociceptive reception system. Two splice variant isoforms have been described.
UniProt Protein Details:	Protein type:Protein kinase, TK; Membrane protein, integral; Oncoprotein; Protein kinase, tyrosine (receptor); Kinase, protein; EC 2.7.10.1; TK group; Trk family Cellular Component: protein complex; cell surface; integral to plasma membrane; early endosome; dendrite; integral to membrane; cell soma; membrane; axon; cytoplasm; late endosome; plasma membrane; cytoplasmic vesicle; receptor complex; endosome Molecular Function:protein homodimerization activity; ephrin receptor binding; nucleotide binding; transmembrane receptor protein tyrosine kinase activity; neurotrophin receptor activity; protein kinase activity; neurotrophin p75 receptor binding; transferase activity; protein binding; nerve growth factor receptor activity; protein-tyrosine kinase activity; transferase activity, transferring phosphorus-containing groups; nerve growth factor binding; kinase activity; kinase binding; ATP binding Biological Process: response to nicotine; axon guidance; mechanoreceptor differentiation; peptidyl-tyrosine phosphorylation; nerve growth factor receptor signaling pathway; multicellular organismal development; protein amino acid autophosphorylation; sensory perception of pain; positive regulation of synaptic transmission, glutamatergic; protein amino acid phosphorylation; activation of NF-kappaB transcription factor; sympathetic nervous system development; learning and/or memory; negative regulation of neuron apoptosis; detection of temperature stimulus involved in sensory perception of pain; response to electrical stimulus; cell differentiation; response to nutrient levels; response to drug; nervous system development; Sertoli cell development; detection of mechanical stimulus involved in sensory perception of pain; positive regulation of angiogenesis; positive regulation of programmed cell death; B cell differentiation; response to activity; phosphorylation; transmembrane receptor protein tyrosine kinase signaling pathway
UniProt Code:	Q3UFB7
NCBI GenInfo Identifier:	126253666
NCBI Gene ID:	18211
NCBI Accession:	Q3UFB7.2
UniProt Related Accession:	Q3UFB7
Molecular Weight:
NCBI Full Name:	High affinity nerve growth factor receptor
NCBI Synonym Full Names:	neurotrophic tyrosine kinase, receptor, type 1
NCBI Official Symbol:	Ntrk1
NCBI Official Synonym Symbols:	Tkr; trk; TrkA; C80751
NCBI Protein Information:	high affinity nerve growth factor receptor
UniProt Protein Name:	High affinity nerve growth factor receptor
UniProt Synonym Protein Names:	Neurotrophic tyrosine kinase receptor type 1
Protein Family:	High affinity nerve growth factor receptor
UniProt Gene Name:	Ntrk1
UniProt Entry Name:	NTRK1_MOUSE

Component	Quantity (96 Assays)	Storage
ELISA Microplate (Dismountable)	8×12 strips	-20°C
Lyophilized Standard	2	-20°C
Sample Diluent	20ml	-20°C
Assay Diluent A	10mL	-20°C
Assay Diluent B	10mL	-20°C
Detection Reagent A	120µL	-20°C
Detection Reagent B	120µL	-20°C
Wash Buffer	30mL	4°C
Substrate	10mL	4°C
Stop Solution	10mL	4°C
Plate Sealer	5	-

Other materials and equipment required:

Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir

*Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.

Step
1.	Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C.
2.	Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform.
3.	Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4.	Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C.
5.	Repeat the wash process for five times as conducted in step 3.
6.	Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction.
7.	Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
8.	Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters.
9.	After experiment, store all reagents according to the specified storage temperature respectively until their expiry.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type	Protocol
Serum	If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma	Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid	Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant	Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates	Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenates	The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates	Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk	Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.