Trk A (Phospho-Tyr680+Tyr681)Colorimetric Cell-Based ELISA Kit (CBCAB00149)

Product Name:	Trk A (Phospho-Tyr680+Tyr681)Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00149
ELISA Type:	Cell-Based
Target:	Trk A (Phospho-Tyr680+Tyr681)
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The Trk A (Phospho-Tyr680+Tyr681) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Trk A protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated Trk A in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on Trk A phosphorylation.

Qualitative determination of Trk A (Phospho-Tyr680+Tyr681) concentration is achieved by an indirect ELISA format. In essence, Trk A (Phospho-Tyr680+Tyr681) is captured by Trk A (Phospho-Tyr680+Tyr681)-specific primary antibodies while the HRP-conjugated secondary antibodies bind the Fc region of the primary antibody. Through this binding, the HRP enzyme conjugated to the secondary antibody can catalyze a colorimetric reaction upon substrate addition. Due to the qualitative nature of the Cell-Based ELISA, multiple normalization methods are needed:

1.	A monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target absorbance values.
2.	Following the colorimetric measurement of HRP activity via substrate addition, the Crystal Violet whole-cell staining method may be used to determine cell density. After staining, the results can be analysed by normalizing the absorbance values to cell amounts, by which the plating difference can be adjusted.

Database Information:	Gene ID: 4914, UniProt ID: P04629, OMIM: 164970/188550/191315/256800, Unigene: Hs.406293
Gene Symbol:	NTRK1
Sub Type:	Phospho

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; Oncoprotein; EC 2.7.10.1; Membrane protein, integral; Protein kinase, tyrosine (receptor); Kinase, protein; TK group; Trk family Chromosomal Location of Human Ortholog: 1q21-q22 Cellular Component: protein complex; cell surface; integral to plasma membrane; late endosome membrane; dendrite; early endosome; cell soma; early endosome membrane; axon; late endosome; plasma membrane; cytoplasmic vesicle; receptor complex; endosome Molecular Function:neurotrophin p75 receptor binding; neurotrophin binding; protein binding; nerve growth factor receptor activity; protein homodimerization activity; ephrin receptor binding; nerve growth factor binding; transmembrane receptor protein tyrosine kinase activity; ATP binding Biological Process: circadian rhythm; axon guidance; mechanoreceptor differentiation; peptidyl-tyrosine phosphorylation; activation of MAPKK activity; nerve growth factor receptor signaling pathway; protein amino acid autophosphorylation; positive regulation of synaptic transmission, glutamatergic; protein amino acid phosphorylation; olfactory nerve development; activation of NF-kappaB transcription factor; negative regulation of cell proliferation; sympathetic nervous system development; response to radiation; learning and/or memory; small GTPase mediated signal transduction; response to axon injury; negative regulation of neuron apoptosis; response to electrical stimulus; detection of temperature stimulus involved in sensory perception of pain; response to hydrostatic pressure; aging; response to drug; response to nutrient levels; phosphoinositide-mediated signaling; adenylate cyclase activation; Sertoli cell development; detection of mechanical stimulus involved in sensory perception of pain; positive regulation of programmed cell death; positive regulation of angiogenesis; response to ethanol; phospholipase C activation; B cell differentiation; Ras protein signal transduction; response to activity; positive regulation of Ras protein signal transduction; transmembrane receptor protein tyrosine kinase signaling pathway Disease: Thyroid Carcinoma, Familial Medullary; Insensitivity To Pain, Congenital, With Anhidrosis
NCBI Summary:	This gene encodes a member of the neurotrophic tyrosine kinase receptor (NTKR) family. This kinase is a membrane-bound receptor that, upon neurotrophin binding, phosphorylates itself and members of the MAPK pathway. The presence of this kinase leads to cell differentiation and may play a role in specifying sensory neuron subtypes. Mutations in this gene have been associated with congenital insensitivity to pain, anhidrosis, self-mutilating behavior, cognitive disability and cancer. Alternate transcriptional splice variants of this gene have been found, but only three have been characterized to date. [provided by RefSeq, Jul 2008]
UniProt Code:	P04629
NCBI GenInfo Identifier:	4585712
NCBI Gene ID:	4914
NCBI Accession:	NP_002520
UniProt Related Accession:	P04629
Molecular Weight:	87.511
NCBI Full Name:	high affinity nerve growth factor receptor isoform 2
NCBI Synonym Full Names:	neurotrophic receptor tyrosine kinase 1
NCBI Official Symbol:	NTRK1
NCBI Official Synonym Symbols:	MTC; TRK; TRK1; TRKA; Trk-A; p140-TrkA
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; TRK1-transforming tyrosine kinase protein; Tropomyosin-related kinase A; Tyrosine kinase receptor; Tyrosine kinase receptor A; Trk-A; gp140trk; p140-TrkA
Protein Family:	Trk system potassium uptake protein
UniProt Gene Name:	NTRK1
UniProt Entry Name:	NTRK1_HUMAN

Component	Quantity
96-Well Cell Culture Clear-Bottom Microplate	2 plates
10X TBS	24 mL
Quenching Buffer	24 mL
Blocking Buffer	50 mL
15X Wash Buffer	50 mL
Primary Antibody Diluent	12 mL
100x Anti-Phospho Target Antibody	60 µL
100x Anti-Target Antibody	60 µL
Anti-GAPDH Antibody	60 µL
HRP-Conjugated Anti-Rabbit IgG Antibody	12 mL
HRP-Conjugated Anti-Mouse IgG Antibody	12 mL
SDS Solution	12 mL
Stop Solution	24 mL
Ready-to-Use Substrate	12 mL
Crystal Violet Solution	12 mL
Adhesive Plate Seals	2 seals

The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:

Microplate reader able to measure absorbance at 450 nm and/or 595 nm for Crystal Violet Cell Staining (Optional)
Micropipettes with capability of measuring volumes ranging from 1 µL to 1 ml
37% formaldehyde (Sigma Cat# F-8775) or formaldehyde from other sources
Squirt bottle, manifold dispenser, multichannel pipette reservoir or automated microplate washer
Graph paper or computer software capable of generating or displaying logarithmic functions
Absorbent papers or vacuum aspirator
Test tubes or microfuge tubes capable of storing ≥1 ml
Poly-L-Lysine (Sigma Cat# P4832 for suspension cells)
Orbital shaker (optional)
Deionized or sterile water

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

Step	Procedure
1.	Seed 200 µL of 20,000 adherent cells in culture medium in each well of a 96-well plate. The plates included in the kit are sterile and treated for cell culture. For suspension cells and loosely attached cells, coat the plates with 100 µL of 10 µg/ml Poly-L-Lysine (not included) to each well of a 96-well plate for 30 minutes at 37°C prior to adding cells.
2.	Incubate the cells for overnight at 37°C, 5% CO2.
3.	Treat the cells as desired.
4.	Remove the cell culture medium and rinse with 200 µL of 1x TBS, twice.
5.	Fix the cells by incubating with 100 µL of Fixing Solution for 20 minutes at room temperature. The 4% formaldehyde is used for adherent cells and 8% formaldehyde is used for suspension cells and loosely attached cells.
6.	Remove the Fixing Solution and wash the plate 3 times with 200 µL 1x Wash Buffer for five minutes each time with gentle shaking on the orbital shaker. The plate can be stored at 4°C for a week.
7.	Add 100 µL of Quenching Buffer and incubate for 20 minutes at room temperature.
8.	Wash the plate 3 times with 1x Wash Buffer for 5 minutes each time.
9.	Add 200 µL of Blocking Buffer and incubate for 1 hour at room temperature.
10.	Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
11.	Add 50 µL of 1x primary antibodies Anti-Trk A (Phospho-Tyr680+Tyr681) Antibody, Anti-Trk A Antibody and/or Anti-GAPDH Antibody) to the corresponding wells, cover with Parafilm and incubate for 16 hours (overnight) at 4°C. If the target expression is known to be high, incubate for 2 hours at room temperature.
12.	Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
13.	Add 50 µL of 1x secondary antibodies (HRP-Conjugated AntiRabbit IgG Antibody or HRP-Conjugated Anti-Mouse IgG Antibody) to corresponding wells and incubate for 1.5 hours at room temperature.
14.	Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
15.	Add 50 µL of Ready-to-Use Substrate to each well and incubate for 30 minutes at room temperature in the dark.
16.	Add 50 µL of Stop Solution to each well and read OD at 450 nm immediately using the microplate reader.

(Additional Crystal Violet staining may be performed if desired – details of this may be found in the kit technical manual.)