Human EPHB2/EPH Receptor B2 ELISA Kit- High Sensitivity

Product Name:	Human EPHB2 / EPH Receptor B2 ELISA Kit
Product Code:	HUFI02425
Size:	96 Assays
Alias:	ERK, Cek5, Drt, Hek5, Nuk, Qek2, Sek3, Tyro5, CAPB, DRTEphB2, EC 2.7.10, EC 2.7.10.1, EK5, elk-related tyrosine kinase, EPH receptor B2, eph tyrosine kinase 3, EPH-like kinase 5, ephrin type-B receptor 2, EPHT3MGC87492, EPTH3, ERKHek5, HEK5, hEK5, PCBC, protein-tyrosine kinase HEK5, Renal carcinoma antigen NY-REN-47, Tyro5, TYRO5, Tyrosine-protein kinase receptor EPH-3, Tyrosine-protein kinase TYRO5
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human ERK concentrations in serum plasma and other biological fluids.
Sensitivity:	0.375ng/ml
Range:	0.625-40ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

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

Matrix	Recovery range(%)	Average(%)
serum(n=5)	90-104	98
EDTA plasma(n=5)	85-103	91
UFH plasma(n=5)	90-99	93

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human ERK 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)	88-95%	97-105%	93-105%
EDTA plasma(n=5)	92-101%	84-99%	83-100%
UFH plasma(n=5)	80-95%	80-98%	81-98%

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

P29323

UniProt Protein Function:	EphB2: a receptor tyrosine kinase of the Eph family. A receptor for ephrin-B family members. Activated EphB2 recruits RasGAP, down-regulating the Ras-Erk signaling axis and neurite retraction. The Eph receptor tyrosine kinase family, the largest in the tyrosine kinase group, has fourteen members. They bind membrane-anchored ligands, ephrins, at sites of cell-cell contact, regulating the repulsion and adhesion of cells that underlie the establishment, maintenance, and remodeling of patterns of cellular organization. Eph signals are particularly important in regulating cell adhesion and cell migration during development, axon guidance, homeostasis and disease. EphA receptors bind to GPI-anchored ephrin-A ligands, while EphB receptors bind to ephrin-B proteins that have a transmembrane and cytoplasmic domain. Interactions between EphB receptor kinases and ephrin-B proteins transduce signals bidirectionally, signaling to both interacting cell types. Eph receptors and ephrins also regulate the adhesion of endothelial cells and are required for the remodeling of blood vessels. The ligand-activated form of EphB2 interacts with multiple proteins, including GTPase-activating protein (RASGAP) through its SH2 domain. Point mutations seen in prostate cancer. Overexpressed and required for migration of glioblastoma. Overexpressed and correlated with poor survival in breast cancer. Overexpression and loss of heterozygosity seen in colorectal cancers. Target for immunoconjugate drug therapy.Three splice-variant isoforms have been described.
UniProt Protein Details:	Protein type:Tumor suppressor; Protein kinase, TK; Protein kinase, tyrosine (receptor); Kinase, protein; Membrane protein, integral; EC 2.7.10.1; TK group; Eph family Chromosomal Location of Human Ortholog: 1p36.1-p35 Cellular Component: axon; cytosol; dendrite; extracellular region; integral to plasma membrane; plasma membrane Molecular Function:protein binding; protein-tyrosine kinase activity; transmembrane-ephrin receptor activity Biological Process: angiogenesis; axon guidance; axonal fasciculation; corpus callosum development; ephrin receptor signaling pathway; inner ear morphogenesis; nervous system development; palate development; peptidyl-tyrosine phosphorylation; phosphorylation; positive regulation of synaptogenesis; regulation of body fluid levels; urogenital system development Disease: Prostate Cancer/brain Cancer Susceptibility
NCBI Summary:	This gene encodes a member of the Eph receptor family of receptor tyrosine kinase transmembrane glycoproteins. These receptors are composed of an N-terminal glycosylated ligand-binding domain, a transmembrane region and an intracellular kinase domain. They bind ligands called ephrins and are involved in diverse cellular processes including motility, division, and differentiation. A distinguishing characteristic of Eph-ephrin signaling is that both receptors and ligands are competent to transduce a signaling cascade, resulting in bidirectional signaling. This protein belongs to a subgroup of the Eph receptors called EphB. Proteins of this subgroup are distinguished from other members of the family by sequence homology and preferential binding affinity for membrane-bound ephrin-B ligands. Allelic variants are associated with prostate and brain cancer susceptibility. Alternative splicing results in multiple transcript variants. [provided by RefSeq, May 2015]
UniProt Code:	P29323
NCBI GenInfo Identifier:	76803654
NCBI Gene ID:	2048
NCBI Accession:	P29323.5
UniProt Secondary Accession:	P29323,O43477, Q5T0U6, Q5T0U7, Q5T0U8,
UniProt Related Accession:	P29323
Molecular Weight:	110,030 Da
NCBI Full Name:	Ephrin type-B receptor 2
NCBI Synonym Full Names:	EPH receptor B2
NCBI Official Symbol:	EPHB2
NCBI Official Synonym Symbols:	DRT; EK5; ERK; CAPB; Hek5; PCBC; EPHT3; Tyro5
NCBI Protein Information:	ephrin type-B receptor 2
UniProt Protein Name:	Ephrin type-B receptor 2
UniProt Synonym Protein Names:	Developmentally-regulated Eph-related tyrosine kinase; ELK-related tyrosine kinase; EPH tyrosine kinase 3; EPH-like kinase 5; EK5; hEK5; Renal carcinoma antigen NY-REN-47; Tyrosine-protein kinase TYRO5; Tyrosine-protein kinase receptor EPH-3
Protein Family:	Ephrin type-B receptor
UniProt Gene Name:	EPHB2
UniProt Entry Name:	EPHB2_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 Âµl 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 Âµl 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.

Human EPHB2 / EPH Receptor B2 ELISA Kit (HUFI02425)

Description