Description
Human Angiopoietin-1 receptor (TEK) ELISA Kit
The Human Angiopoietin-1 receptor (TEK) ELISA Kit is specifically designed for the accurate measurement of Angiopoietin-1 receptor levels in human samples such as serum, plasma, and cell culture supernatants. This kit is highly sensitive and specific, ensuring reliable and reproducible results for a variety of research applications.Angiopoietin-1 receptor, also known as TEK, is a key player in angiogenesis, the process of blood vessel formation.
This receptor is crucial in regulating vascular development and stability, making it a valuable biomarker for investigating diseases such as cancer, cardiovascular disorders, and neurodegenerative conditions. By accurately measuring Angiopoietin-1 receptor levels, researchers can gain valuable insights into the underlying mechanisms of these diseases and potentially identify new therapeutic targets.
| Product Name: | Human Angiopoietin-1 receptor (TEK) ELISA Kit |
| SKU: | HUEB0151 |
| Size: | 96T |
| Target: | Human Angiopoietin-1 receptor (TEK) |
| Synonyms: | Endothelial tyrosine kinase, Tunica interna endothelial cell kinase, Tyrosine kinase with Ig and EGF homology domains-2, Tyrosine-protein kinase receptor TEK, Tyrosine-protein kinase receptor TIE-2, p140 TEK, hTIE2, CD202b, TIE2, VMCM, VMCM1 |
| Assay Type: | Sandwich |
| Detection Method: | ELISA |
| Reactivity: | Human |
| Detection Range: | 0.156-10ng/mL |
| Sensitivity: | 0.039ng/mL |
| Intra CV: | 4.6% | ||||||||||||||||||||
| Inter CV: | 9.3% | ||||||||||||||||||||
| Linearity: |
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| Recovery: |
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| Function: | Tyrosine-protein kinase that acts as cell-surface receptor for ANGPT1, ANGPT2 and ANGPT4 and regulates angiogenesis, endothelial cell survival, proliferation, migration, adhesion and cell spreading, reorganization of the actin cytoskeleton, but also maintenance of vascular quiescence. Has anti-inflammatory effects by preventing the leakage of proinflammatory plasma proteins and leukocytes from blood vessels. Required for normal angiogenesis and heart development during embryogenesis. Required for post-natal hematopoiesis. After birth, activates or inhibits angiogenesis, depending on the context. Inhibits angiogenesis and promotes vascular stability in quiescent vessels, where endothelial cells have tight contacts. In quiescent vessels, ANGPT1 oligomers recruit TEK to cell-cell contacts, forming complexes with TEK molecules from adjoining cells, and this leads to preferential activation of phosphatidylinositol 3-kinase and the AKT1 signaling cascades. In migrating endothelial cells that lack cell-cell adhesions, ANGT1 recruits TEK to contacts with the extracellular matrix, leading to the formation of focal adhesion complexes, activation of PTK2/FAK and of the downstream kinases MAPK1/ERK2 and MAPK3/ERK1, and ultimately to the stimulation of sprouting angiogenesis. ANGPT1 signaling triggers receptor dimerization and autophosphorylation at specific tyrosine residues that then serve as binding sites for scaffold proteins and effectors. Signaling is modulated by ANGPT2 that has lower affinity for TEK, can promote TEK autophosphorylation in the absence of ANGPT1, but inhibits ANGPT1-mediated signaling by competing for the same binding site. Signaling is also modulated by formation of heterodimers with TIE1, and by proteolytic processing that gives rise to a soluble TEK extracellular domain. The soluble extracellular domain modulates signaling by functioning as decoy receptor for angiopoietins. TEK phosphorylates DOK2, GRB7, GRB14, PIK3R1; SHC1 and TIE1. |
| Uniprot: | Q02763 |
| Sample Type: | Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids |
| Specificity: | Natural and recombinant human Angiopoietin-1 receptor |
| Sub Unit: | Homodimer. Heterodimer with TIE1. Interacts with ANGPT1, ANGPT2 and ANGPT4. At cell-cell contacts in quiescent cells, forms a signaling complex composed of ANGPT1 plus TEK molecules from two adjoining cells. In the absence of endothelial cell-cell contacts, interaction with ANGPT1 mediates contacts with the extracellular matrix. Interacts with PTPRB; this promotes endothelial cell-cell adhesion. Interacts with DOK2, GRB2, GRB7, GRB14, PIK3R1 and PTPN11/SHP2. Colocalizes with DOK2 at contacts with the extracellular matrix in migrating cells. Interacts (tyrosine phosphorylated) with TNIP2. Interacts (tyrosine phosphorylated) with SHC1 (via SH2 domain). |
| Research Area: | Development Biology |
| Subcellular Location: | Cell membrane Single-pass type I membrane protein Cell junction Cell junction Focal adhesion Cytoplasm Cytoskeleton Secreted Recruited to cell-cell contacts in quiescent endothelial cells. Colocalizes with the actin cytoskeleton and at actin stress fibers during cell spreading. Recruited to the lower surface of migrating cells, especially the rear end of the cell. Proteolytic processing gives rise to a soluble extracellular domain that is secreted. |
| Storage: | Please see kit components below for exact storage details |
| Note: | For research use only |
| UniProt Protein Function: | TIE2: a receptor tyrosine kinase of the Tie family. Receptor for angiopoietin 1. Expressed almost exclusively in endothelial cells. May constitute the earliest mammalian endothelial cell lineage marker. Appears to be critical for endothelial cell-smooth muscle cell communication in venous morphogenesis. TEK is closely related to the TIE receptor tyrosine kinase. |
| UniProt Protein Details: | Protein type:Protein kinase, TK; Protein kinase, tyrosine (receptor); EC 2.7.10.1; Kinase, protein; Membrane protein, integral; TK group; Tie family Chromosomal Location of Human Ortholog: 9p21 Cellular Component: microvillus; focal adhesion; cell surface; basolateral plasma membrane; integral to plasma membrane; extracellular region; actin filament; intercellular junction; lipid raft; perinuclear region of cytoplasm; apical plasma membrane; stress fiber; basal plasma membrane; plasma membrane Molecular Function:protein binding; growth factor binding; protein-tyrosine kinase activity; receptor activity; transmembrane receptor protein tyrosine kinase activity; ATP binding; protein kinase activity Biological Process: response to peptide hormone stimulus; peptidyl-tyrosine phosphorylation; response to cAMP; heart development; protein amino acid autophosphorylation; cell-matrix adhesion; positive regulation of cytokine secretion during immune response; signal transduction; cell-cell adhesion; cell-cell signaling; positive regulation of focal adhesion formation; angiogenesis; endochondral ossification; organ regeneration; Tie receptor signaling pathway; regulation of establishment and/or maintenance of cell polarity; positive regulation of phosphoinositide 3-kinase activity; positive regulation of peptidyl-serine phosphorylation; protein oligomerization; positive regulation of phosphoinositide 3-kinase cascade; positive regulation of protein kinase B signaling cascade; negative regulation of angiogenesis; positive regulation of angiogenesis; positive regulation of protein import into nucleus; response to estrogen stimulus; negative regulation of inflammatory response; endothelial cell proliferation; response to hypoxia; positive regulation of endothelial cell proliferation; regulation of vascular permeability; sprouting angiogenesis; positive regulation of protein amino acid phosphorylation; blood coagulation; leukocyte migration; transmembrane receptor protein tyrosine kinase signaling pathway; negative regulation of apoptosis Disease: Venous Malformations, Multiple Cutaneous And Mucosal |
| NCBI Summary: | The TEK receptor tyrosine kinase is expressed almost exclusively in endothelial cells in mice, rats, and humans. This receptor possesses a unique extracellular domain containing 2 immunoglobulin-like loops separated by 3 epidermal growth factor-like repeats that are connected to 3 fibronectin type III-like repeats. The ligand for the receptor is angiopoietin-1. Defects in TEK are associated with inherited venous malformations; the TEK signaling pathway appears to be critical for endothelial cell-smooth muscle cell communication in venous morphogenesis. TEK is closely related to the TIE receptor tyrosine kinase. [provided by RefSeq, Jul 2008] |
| UniProt Code: | Q02763 |
| NCBI GenInfo Identifier: | 218511853 |
| NCBI Gene ID: | 7010 |
| NCBI Accession: | Q02763.2 |
| UniProt Secondary Accession: | Q02763,Q5TCU2, Q8IV34, A8K6W0, B4DH20, B4DHD3, D3DRK5 E7EWI2, |
| UniProt Related Accession: | Q02763 |
| Molecular Weight: | 60.2kD |
| NCBI Full Name: | Angiopoietin-1 receptor |
| NCBI Synonym Full Names: | TEK tyrosine kinase, endothelial |
| NCBI Official Symbol: | TEK |
| NCBI Official Synonym Symbols: | TIE2; VMCM; TIE-2; VMCM1; CD202B |
| NCBI Protein Information: | angiopoietin-1 receptor; hTIE2; p140 TEK; soluble TIE2 variant 1; soluble TIE2 variant 2; endothelial tyrosine kinase; tyrosine-protein kinase receptor TEK; tunica interna endothelial cell kinase; tyrosine-protein kinase receptor TIE-2; tyrosine kinase with Ig and EGF homology domains-2 |
| UniProt Protein Name: | Angiopoietin-1 receptor |
| UniProt Synonym Protein Names: | Endothelial tyrosine kinase; Tunica interna endothelial cell kinase; Tyrosine kinase with Ig and EGF homology domains-2; Tyrosine-protein kinase receptor TEK; Tyrosine-protein kinase receptor TIE-2; hTIE2; p140 TEK |
| Protein Family: | Angiopoietin-1 receptor |
| UniProt Gene Name: | TEK |
| UniProt Entry Name: | TIE2_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. |
