Description
Human Ephrin type-A receptor 3 (EPHA3) ELISA Kit
The Human Ephrin Type-A Receptor 3 (EPHA3) ELISA Kit is a powerful tool for the precise measurement of EPHA3 levels in human samples including serum, plasma, and cell culture supernatants. With its exceptional sensitivity and specificity, this kit delivers accurate and consistent results, making it an invaluable asset for a wide array of research endeavors.EPHA3 is a key receptor involved in various cellular processes, particularly in mediating cell-cell interactions and influencing tissue development. Dysregulation of EPHA3 has been implicated in a range of diseases including cancer, neurological disorders, and cardiovascular conditions, underscoring its importance as a biomarker for disease progression and potential therapeutic targets.
With the Human EPHA3 ELISA Kit, researchers can uncover valuable insights into the role of EPHA3 in disease pathogenesis and explore its potential as a target for novel therapeutic interventions. Trust in this kit to provide reliable data and facilitate groundbreaking discoveries in the field of molecular biology and medicine.
| Product Name: | Human Ephrin type-A receptor 3 (EPHA3) ELISA Kit |
| SKU: | HUEB1119 |
| Size: | 96T |
| Target: | Human Ephrin type-A receptor 3 (EPHA3) |
| Synonyms: | EPH-like kinase 4, HEK, Tyrosine-protein kinase TYRO4, Tyrosine-protein kinase receptor ETK1, EK4, Human embryo kinase, Eph-like tyrosine kinase 1, ETK, ETK1, HEK, TYRO4 |
| Assay Type: | Sandwich |
| Detection Method: | ELISA |
| Reactivity: | Human |
| Detection Range: | 0.312-20ng/mL |
| Sensitivity: | 0.11ng/mL |
| Intra CV: | 5.1% | ||||||||||||||||||||
| Inter CV: | 8.3% | ||||||||||||||||||||
| Linearity: |
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| Recovery: |
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| Function: | Receptor tyrosine kinase which binds promiscuously membrane-bound ephrin 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. Highly promiscuous for ephrin-A ligands it binds preferentially EFNA5. Upon activation by EFNA5 regulates cell-cell adhesion, cytoskeletal organization and cell migration. Plays a role in cardiac cells migration and differentiation and regulates the formation of the atrioventricular canal and septum during development probably through activation by EFNA1. Involved in the retinotectal mapping of neurons. May also control the segregation but not the guidance of motor and sensory axons during neuromuscular circuit development. |
| Uniprot: | P29320 |
| Sample Type: | Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids |
| Specificity: | Natural and recombinant human Ephrin type-A receptor 3 |
| Sub Unit: | Heterotetramer upon binding of the ligand. The heterotetramer is composed of an ephrin dimer and a receptor dimer. Oligomerization is probably required to induce biological responses. Forms a ternary EFNA5-EPHA3-ADAM10 complex mediating EFNA5 extracellular domain shedding by ADAM10 which regulates the EFNA5-EPHA3 complex internalization and function. Interacts with NCK1 (via SH2 domain); mediates EFNA5-EPHA3 signaling (By similarity). Interacts (phosphorylated) with PTPN1; dephosphorylates EPHA3 and may regulate its trafficking and function. Interacts (phosphorylated) with CRK; mediates EFNA5-EPHA3 signaling through RHOA GTPase activation. |
| Research Area: | Signal Transduction |
| Subcellular Location: | Isoform 2 Secreted |
| Storage: | Please see kit components below for exact storage details |
| Note: | For research use only |
| UniProt Protein Function: | EphA3: a receptor tyrosine kinase of the Eph family. Receptor for members of the ephrin-A family: binds to ephrin-A2, -A3, -A4 and -A5The 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. Two point mutations seen in a survey of colorectal tumors. Soluble receptors reduce tumor growth and angiogenesis in mouse models. Two alternatively spliced isoforms of EphA3 have been described. |
| UniProt Protein Details: | Protein type:EC 2.7.10.1; Protein kinase, tyrosine (receptor); Protein kinase, TK; Membrane protein, integral; Kinase, protein; TK group; Eph family Chromosomal Location of Human Ortholog: 3p11.2 Cellular Component: early endosome; extracellular region; integral to plasma membrane; plasma membrane Molecular Function:ATP binding; GPI-linked ephrin receptor activity; protein binding Biological Process: cell adhesion; cell migration; ephrin receptor signaling pathway; peptidyl-tyrosine phosphorylation; regulation of actin cytoskeleton organization and biogenesis; regulation of focal adhesion formation; regulation of GTPase activity |
| NCBI Summary: | This gene belongs to the ephrin receptor subfamily of the protein-tyrosine kinase family. EPH and EPH-related receptors have been implicated in mediating developmental events, particularly in the nervous system. Receptors in the EPH subfamily typically have a single kinase domain and an extracellular region containing a Cys-rich domain and 2 fibronectin type III repeats. The ephrin receptors are divided into 2 groups based on the similarity of their extracellular domain sequences and their affinities for binding ephrin-A and ephrin-B ligands. This gene encodes a protein that binds ephrin-A ligands. Two alternatively spliced transcript variants have been described for this gene. [provided by RefSeq, Jul 2008] |
| UniProt Code: | P29320 |
| NCBI GenInfo Identifier: | 116241351 |
| NCBI Gene ID: | 2042 |
| NCBI Accession: | P29320.2 |
| UniProt Secondary Accession: | P29320,Q9H2V3, Q9H2V4, |
| UniProt Related Accession: | P29320 |
| Molecular Weight: | 60,946 Da |
| NCBI Full Name: | Ephrin type-A receptor 3 |
| NCBI Synonym Full Names: | EPH receptor A3 |
| NCBI Official Symbol: | EPHA3 |
| NCBI Official Synonym Symbols: | EK4; ETK; HEK; ETK1; HEK4; TYRO4 |
| NCBI Protein Information: | ephrin type-A receptor 3 |
| UniProt Protein Name: | Ephrin type-A receptor 3 |
| UniProt Synonym Protein Names: | EPH-like kinase 4; EK4; hEK4; HEK; Human embryo kinase; Tyrosine-protein kinase TYRO4; Tyrosine-protein kinase receptor ETK1; Eph-like tyrosine kinase 1 |
| Protein Family: | Ephrin type-A receptor |
| UniProt Gene Name: | EPHA3 |
| UniProt Entry Name: | EPHA3_HUMAN |
| Component | Quantity (96 Assays) | Storage |
| ELISA Microplate (Dismountable) | 8×12 strips | -20°C |
| Lyophilized Standard | 2 | -20°C |
| Sample Diluent | 20ml | -20°C |
| Assay Diluent A | 10mL | -20°C |
| Assay Diluent B | 10mL | -20°C |
| Detection Reagent A | 120µL | -20°C |
| Detection Reagent B | 120µL | -20°C |
| Wash Buffer | 30mL | 4°C |
| Substrate | 10mL | 4°C |
| Stop Solution | 10mL | 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
*Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.
| Step | |
| 1. | Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C. |
| 2. | Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform. |
| 3. | Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper. |
| 4. | Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C. |
| 5. | Repeat the wash process for five times as conducted in step 3. |
| 6. | Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction. |
| 7. | Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing. |
| 8. | Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters. |
| 9. | After experiment, store all reagents according to the specified storage temperature respectively until their expiry. |
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. |
