Rat TrkB/NTRK2 ELISA Kit (RTFI01001)

Product Name:	Rat TrkB/NTRK2 ELISA Kit
Product Code:	RTFI01001
Size:	96 Assays
Target:	Rat NTRK2
Alias:	NTRK2, BDNF, NT-3 growth factors receptor, GP145-TrkB, neurotrophic tyrosine kinase, receptor, type 2, trk-B, Trk-B, TrkB tyrosine kinase, TRKBBDNF-tropomyosine receptor kinase B, tyrosine kinase receptor B
Reactivity:	Rat
Detection Method:	Sandwich ELISA, Double Antibody
Sensitivity:	56.25pg/ml
Range:	93.75-6000pg/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Rat NTRK2 and the recovery rates were calculated by comparing the measured value to the expected amount of Rat NTRK2 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	91-104	99
EDTA plasma(n=5)	91-103	96
UFH plasma(n=5)	86-104	94

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Rat NTRK2 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample	1:2	1:4	1:8
serum(n=5)	92-105%	89-99%	95-104%
EDTA plasma(n=5)	83-97%	83-98%	84-98%
UFH plasma(n=5)	80-99%	80-87%	80-99%

Intra-Assay:

CV <8%

Inter-Assay:

CV <10%

Uniprot:

Q63604

UniProt Protein Function:	TrkB: a receptor tyrosine kinase of the Trk family. Receptor for brain-derived neurotrophic factor (BDNF), neurotrophin-3, -4 and -5 but not nerve growth factor (NGF). Involved in the development and maintenance of the nervous system. Three splice variant isoforms have been described.
UniProt Protein Details:	Protein type:Membrane protein, integral; EC 2.7.10.1; Protein kinase, tyrosine (receptor); Protein kinase, TK; Kinase, protein; TK group; Trk family Chromosomal Location of Human Ortholog: 9q22.1 Cellular Component: postsynaptic membrane; cell surface; growth cone; rough endoplasmic reticulum; integral to plasma membrane; dendritic spine; presynaptic active zone; excitatory synapse; terminal button; perikaryon; endosome membrane; cytosol; receptor complex Molecular Function:brain-derived neurotrophic factor receptor activity; neurotrophin binding; protein homodimerization activity; ephrin receptor binding; brain-derived neurotrophic factor binding; ATP binding Biological Process: circadian rhythm; mechanoreceptor differentiation; retinal rod cell development; peptidyl-tyrosine phosphorylation; nerve growth factor receptor signaling pathway; protein amino acid autophosphorylation; regulation of dendrite development; neuron migration; regulation of neurotransmitter secretion; positive regulation of synaptic transmission, glutamatergic; neuron differentiation; positive regulation of MAPKKK cascade; positive regulation of cell proliferation; oligodendrocyte differentiation; feeding behavior; negative regulation of neuron apoptosis; inflammatory response; vasculogenesis; neuromuscular junction development; central nervous system neuron development; aging; response to light stimulus; brain-derived neurotrophic factor receptor signaling pathway; adenylate cyclase activation; regulation of protein kinase B signaling cascade; learning; positive regulation of peptidyl-serine phosphorylation; positive regulation of phosphoinositide 3-kinase cascade; peripheral nervous system neuron development; long-term memory; glutamate secretion; positive regulation of axonogenesis; cerebral cortex development; transmembrane receptor protein tyrosine kinase signaling pathway Disease: Obesity, Hyperphagia, And Developmental Delay
NCBI Summary:	This gene encodes a member of the neurotrophic tyrosine receptor kinase (NTRK) family. This kinase is a membrane-bound receptor that, upon neurotrophin binding, phosphorylates itself and members of the MAPK pathway. Signalling through this kinase leads to cell differentiation. Mutations in this gene have been associated with obesity and mood disorders. Alternate transcriptional splice variants encoding different isoforms have been found for this gene. [provided by RefSeq, Jul 2008]
UniProt Code:	Q63604
NCBI GenInfo Identifier:	55956790
NCBI Gene ID:	4915
NCBI Accession:	NP_001007098.1
UniProt Secondary Accession:	Q63604,P15209, Q63604,
UniProt Related Accession:	Q16620
Molecular Weight:	91,999 Da
NCBI Full Name:	BDNF/NT-3 growth factors receptor isoform b
NCBI Synonym Full Names:	neurotrophic tyrosine kinase, receptor, type 2
NCBI Official Symbol:	NTRK2
NCBI Official Synonym Symbols:	TRKB; trk-B; GP145-TrkB
NCBI Protein Information:	BDNF/NT-3 growth factors receptor; trkB tyrosine kinase; tyrosine kinase receptor B; tropomyosin-related kinase B; BDNF-tropomyosine receptor kinase B
UniProt Protein Name:	BDNF/NT-3 growth factors receptor
UniProt Synonym Protein Names:	GP145-TrkB; Trk-B; Neurotrophic tyrosine kinase receptor type 2; TrkB tyrosine kinase; Tropomyosin-related kinase B
UniProt Gene Name:	NTRK2
UniProt Entry Name:	NTRK2_HUMAN

Step	Procedure
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 clotovernight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Removeserum 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 anti-coagulant. 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 & homogenizein 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.