Human NGFR ELISA Kit (HUFI03107)- High Sensitivity

Product Name:	Human NGFR (p75 neurotrophin receptor) ELISA Kit
Product Code:	HUFI03107
Size:	96 Assays
Alias:	NGFR
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human NGFR concentrations in serum plasma and other biological fluids.
Sensitivity:	0.938ng/ml
Range:	1.563-100ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:	Matrices listed below were spiked with certain level of Human NGFR and the recovery rates were calculated by comparing the measured value to the expected amount of Human NGFR in samples. Enquire for more information.
Linearity:	The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human NGFR and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected. Enquire for more information.
CV(%):	Intra-Assay: CV<8% Inter-Assay: CV<10%

Component	Quantity	Storage
ELISA Microplate (Dismountable)	8×12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/-20°C
Sample/Standard Dilution Buffer	20ml	4°C
Biotin-labeled Antibody(Concentrated)	120ul	4°C (Protect from light)
Antibody Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate(SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protect from light)
Stop Solution	10ml	4°C
Wash Buffer(25X)	30ml	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

Uniprot

P08138

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

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

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Step	Protocol
1.	Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.	Aliquot 0.1ml standard solutions into the standard wells.
3.	Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.	Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.	Seal the plate with a cover and incubate at 37 °C for 90 min.
6.	Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.	Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.	Seal the plate with a cover and incubate at 37°C for 60 min.
9.	Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.	Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.	Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.	Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.	Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14.	Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

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.