Human EPHB2 / EPH Receptor B2 ELISA Kit

(No reviews yet) Write a Review
SKU:
HUFI02425
€599

Description

ELISA Kit Technical ManualELISA Kit Technical ManualMSDS

Human EPHB2 / EPH Receptor B2 ELISA Kit - Information

The ELISA Genie EPHB2 / EPH Receptor B2 ELISA Kit can assay for EPHB2 / EPH Receptor B2 in the following samples: serum, blood, plasma, cell culture supernatant and other related supernatants and tissues.

How our EPHB2 / EPH Receptor B2 ELISA Kits Work?

The ELISA Genie (enzyme-linked immunosorbent assays) assay kits are designed for the quantitative measurement of analytes in a wide variety of samples. As today's scientists demand high quality consistent data for high impact journals, ELISA Genie have developed our range of sensitive, fast and reliable ELISA kit assays to meet and exceed those demands. Our assay kits use a quantitative sandwich ELISA technique and each kit comes with highly specific antibodies pre-coated onto a 96-well microtiter plate.

At ELISA Genie we understand the need for speed! Therefore, we have developed an ultra-fast protocol meaning you achieve your results rapidly. So, once you have prepared and plated your samples, blanks and standards, you simply incubate with a highly specific biotin-conjugated primary antibody and Avidin conjugated to Horseradish Peroxidase (HRP) and incubate for the appropriate length of time. After washing the plate according to the protocol and addition of the TMB (3,3',5,5'-Tetramethylbenzidine) solution, the appearance of a blue colour should be detected due to an enzymatic reaction catalysed by HRP. Next step is the addition of the Stop Solution which terminates the HRP reaction and the blue colour turns yellow with the signal intensity measured on a plate reader at 450nm. The amount of bound EPHB2 / EPH Receptor B2 is proportional to the signal generated by the reaction meaning the kit assay gives you a quantitative measurement of the analyte in your samples.

Human EPHB2 / EPH Receptor B2 ELISA Kit - Data

Description

Receptor tyrosine kinase which binds promiscuously transmembrane ephrin-B family ligands residing on adjacent cells, leading to contact-dependent bidirectional signaling into neighboring cells. The signaling pathway downstream of the receptor is referred to as forward signaling while the signaling pathway downstream of the ephrin ligand is referred to as reverse signaling. Functions in axon guidance during development. Involved in the guidance of commissural axons, that form a major interhemispheric connection between the 2 temporal lobes of the cerebral cortex. Also involved in guidance of contralateral inner ear efferent growth cones at the midline and of retinal ganglion cell axons to the optic disk. In addition to axon guidance, also regulates dendritic spines development and maturation and stimulates the formation of excitatory synapses. Upon activation by EFNB1, abolishes the ARHGEF15-mediated negative regulation on excitatory synapse formation. Controls other aspects of development including angiogenesis, palate development and in inner ear development through regulation of endolymph production. Forward and reverse signaling through the EFNB2/EPHB2 complex regulate movement and adhesion of cells that tubularize the urethra and septate the cloaca. May function as a tumor suppressor.

Post-Translational Modification

Uniprot ID P29323
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 ERK concentrations in serum plasma and other biological fluids.

Size

96T

Range

0.625-40ng/ml

Sensitivity

< 0.375ng/ml

Storage

4'C for 6 months

Recovery

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

Matrix Recovery range(%) Average(%)
serum(n=5) 88-105 100
EDTA plasma(n=5) 85-98 92
UFH plasma(n=5) 91-103 96
Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of 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 1:16
serum(n=5) 85-95% 88-105% 86-104% 86-97%
EDTA plasma(n=5) 82-96% 83-99% 82-90% 82-97%
UFH plasma(n=5) 80-94% 82-97% 83-89% 85-99%
CV(%)

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Note

For Research Use Only

Human EPHB2 / EPH Receptor B2 ELISA Kit Protocol

The below protocol is a sample protocol for Human EPHB2 / EPH Receptor B2 ELISA Kit using a biotinylated detection antibody and streptavidin-HRP. Sandwich ELISAs allow for the detection and quantification of an analyte in a sample by using known analyte concentrations as standards and plotting absorbance of known concentrations vs known standard concentrations. This allows the researcher to calculate the amount of Human EPHB2 / EPH Receptor B2 present in their sample.

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.

Sandwich Protocol

Sandwich ELISA Protocol

Kit 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.

Human EPHB2 / EPH Receptor B2 ELISA Kit components

96 Assays

Storage

ELISA Microplate(Dismountable)8×12 strips4°C for 6 months
Lyophilized Standard24°C/-20°C
Sample/Standard Dilution Buffer20ml4°C
Biotin-labeled Antibody(Concentrated)120ul4°C (Protect from light)
Antibody Dilution Buffer10ml4°C
HRP-Streptavidin Conjugate(SABC)120ul4°C (Protect from light)
SABC Dilution Buffer10ml4°C
TMB Substrate10ml4°C (Protect from light)
Stop Solution10ml4°C
Wash Buffer(25X)30ml4°C
Plate Sealer5 -

Other materials and equipment required:

The ELISA Genie Human EPHB2 / EPH Receptor B2 ELISA Kit will require other equipment and materials to carry out the assay. Please see list below for further details.
  • 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

Sample Preparation

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 TypeProtocol

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 Protein Information

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
View AllClose

Additional Information

Reactivity:
Human
ELISA Type:
Sandwich
View AllClose