TNR16 Colorimetric Cell-Based ELISA (CBCAB00972)

Product Name:	TNR16 Colorimetric Cell-Based ELISA
Product Code:	CBCAB00972
ELISA Type:	Cell-Based
Target:	TNR16
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

The TNR16 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect TNR16 protein expression profile in cells. The kit can be used for measuring the relative amounts of TNR16 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on TNR16.

Qualitative determination of TNR16 concentration is achieved by an indirect ELISA format. In essence, TNR16 is captured by TNR16-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: 4804, UniProt ID: P08138, OMIM: 162010, Unigene: Hs.415768/Hs.681726
Gene Symbol:	NGFR
Sub Type:	None

UniProt Protein Function:	NGFR: Low affinity receptor which can bind to NGF, BDNF, NT-3, and NT-4. Can mediate cell survival as well as cell death of neural cells. Homodimer; disulfide-linked. Interacts with p75NTR- associated cell death executor. Interacts with TRAF2, TRAF4, TRAF6, PTPN13 and RANBP9. Interacts through TRAF6 with SQSTM1 which bridges NGFR to NTRK1. Interacts with BEX1 and NGFRAP1/BEX3. Interacts with KIDINS220 and NTRK1. Can form a ternary complex with NTRK1 and KIDINS220 and this complex is affected by the expression levels of KIDINS220. An increase in KIDINS220 expression leads to a decreased association of NGFR and NTRK1. Interacts with NTRK2; may regulate the ligand specificity of the NTRK2 receptor. Interacts with LINGO1.
UniProt Protein Details:	Protein type:Receptor, misc.; Membrane protein, integral Chromosomal Location of Human Ortholog: 17q21-q22 Cellular Component: cell surface; clathrin-coated endocytic vesicle; cytosol; endosome; external side of plasma membrane; extracellular region; Golgi apparatus; integral to plasma membrane; lipid raft; neuron projection; nuclear envelope; nucleoplasm; nucleus; perikaryon; plasma membrane; rough endoplasmic reticulum Molecular Function:beta-amyloid binding; nerve growth factor binding; neurotrophin TRKA receptor binding; protein binding; Rab GTPase binding; receptor activity; signal transducer activity; transmembrane receptor activity; tumor necrosis factor receptor activity; ubiquitin protein ligase binding Biological Process: axon guidance; central nervous system development; circadian regulation of gene expression; detection of temperature stimulus; glucose homeostasis; hair follicle morphogenesis; immune response; inflammatory response; intracellular protein transport; membrane protein intracellular domain proteolysis; negative regulation of apoptosis; negative regulation of axonogenesis; negative regulation of cell cycle; negative regulation of fibroblast growth factor receptor signaling pathway; negative regulation of hair follicle development; negative regulation of mitochondrial depolarization; negative regulation of neuron apoptosis; nerve development; nerve growth factor receptor signaling pathway; positive regulation of apoptosis; positive regulation of axonogenesis; positive regulation of fibroblast proliferation; positive regulation of MAPKKK cascade; positive regulation of myelination; positive regulation of odontogenesis of dentine-containing teeth; positive regulation of protein kinase B signaling cascade; positive regulation of Rho protein signal transduction; positive regulation of synaptic transmission, cholinergic; positive regulation of synaptic transmission, glutamatergic; regulation of apoptosis; regulation of axonogenesis; regulation of caspase activity; regulation of cell proliferation; response to lipopolysaccharide; response to wounding; sensory perception of pain; tumor necrosis factor-mediated signaling pathway
NCBI Summary:	Nerve growth factor receptor contains an extracellular domain containing four 40-amino acid repeats with 6 cysteine residues at conserved positions followed by a serine/threonine-rich region, a single transmembrane domain, and a 155-amino acid cytoplasmic domain. The cysteine-rich region contains the nerve growth factor binding domain. [provided by RefSeq, Jul 2008]
UniProt Code:	P08138
NCBI GenInfo Identifier:	128156
NCBI Gene ID:	4804
NCBI Accession:	P08138.1
UniProt Secondary Accession:	P08138,B2R961, B4E096,
UniProt Related Accession:	P08138
Molecular Weight:	35,767 Da
NCBI Full Name:	Tumor necrosis factor receptor superfamily member 16
NCBI Synonym Full Names:	nerve growth factor receptor
NCBI Official Symbol:	NGFR
NCBI Official Synonym Symbols:	CD271; p75NTR; TNFRSF16; p75(NTR); Gp80-LNGFR
NCBI Protein Information:	tumor necrosis factor receptor superfamily member 16
UniProt Protein Name:	Tumor necrosis factor receptor superfamily member 16
UniProt Synonym Protein Names:	Gp80-LNGFR; Low affinity neurotrophin receptor p75NTR; Low-affinity nerve growth factor receptor; NGF receptor; p75 ICD; CD_antigen: CD271
Protein Family:	Tumor necrosis factor receptor superfamily
UniProt Gene Name:	NGFR
UniProt Entry Name:	TNR16_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-TNR16 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.)