Mouse TEK / TIE2 ELISA Kit
Mouse TEK / TIE2 ELISA Kit - Information
The ELISA Genie Mouse TEK / TIE2 ELISA Kit can assay for Mouse TEK / TIE2 in the following samples: serum, blood, plasma, cell culture supernatant and other related supernatants and tissues.How our Mouse TEK / TIE2 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 Mouse TEK / TIE2 is proportional to the signal generated by the reaction meaning the kit assay gives you a quantitative measurement of the analyte in your samples.
Mouse TEK / TIE2 ELISA Kit - Data
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.
|Post-Translational Modification|| |
Proteolytic processing leads to the shedding of the extracellular domain (soluble TIE-2 alias sTIE-2). Autophosphorylated on tyrosine residues in response to ligand binding. Autophosphorylation occurs in trans, i.e. one subunit of the dimeric receptor phosphorylates tyrosine residues on the other subunit. Autophosphorylation occurs in a sequential manner, where Tyr-990 in the kinase activation loop is phosphorylated first, followed by autophosphorylation at Tyr-1106 and at additional tyrosine residues. ANGPT1-induced phosphorylation is impaired during hypoxia, due to increased expression of ANGPT2. Phosphorylation is important for interaction with GRB14, PIK3R1 and PTPN11. Phosphorylation at Tyr-1100 is important for interaction with GRB2 and GRB7. Phosphorylation at Tyr-1106 is important for interaction with DOK2 and for coupling to downstream signal transduction pathways in endothelial cells. Dephosphorylated by PTPRB. Ubiquitinated. The phosphorylated receptor is ubiquitinated and internalized, leading to its degradation.
|Detection method|| |
Sandwich ELISA Double Antibody
This immunoassay kit allows for the in vitro quantitative determination of Tek/Tie-2/TEK/CD202B/TIE2/VMCM/VMCM1/Angiopoietin-1 receptor/ANG-R-Tie2/Angiopoietin Receptor Tie2 concentrations in serum plasma and other biological fluids.
4'C for 6 months
Matrices listed below were spiked with certain level of Tek/Tie-2/TEK/CD202B/TIE2/VMCM/VMCM1/Angiopoietin-1 receptor/ANG-R-Tie2/Angiopoietin Receptor Tie2 and the recovery rates were calculated by comparing the measured value to the expected amount of Tek/Tie-2/TEK/CD202B/TIE2/VMCM/VMCM1/Angiopoietin-1 receptor/ANG-R-Tie2/Angiopoietin Receptor Tie2 in samples.
The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Tek/Tie-2/TEK/CD202B/TIE2/VMCM/VMCM1/Angiopoietin-1 receptor/ANG-R-Tie2/Angiopoietin Receptor Tie2 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
For Research Use Only
Mouse TEK / TIE2 ELISA Kit Protocol
The below protocol is a sample protocol for Mouse TEK / TIE2 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 Mouse TEK / TIE2 present in their sample.
Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37 Â°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.
|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 Â°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 37Â°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 37Â°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 37Â°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.|
Mouse TEK / TIE2 ELISA Kit components
|ELISA Microplate(Dismountable)||8×12 strips||4°C for 6 months|
|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)|
Other materials and equipment required:The ELISA Genie Mouse TEK / TIE2 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
- Deionized or distilled water
- Absorbent paper
- Buffer resevoir
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.
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.
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.
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.
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.
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.
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.
Mouse TEK / TIE2 ELISA Kit Protein Information
|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:Kinase, protein; Membrane protein, integral; EC 188.8.131.52; Protein kinase, tyrosine (receptor); Protein kinase, TK; TK group; Tie family
Cellular Component: microvillus; cell surface; integral to plasma membrane; basolateral plasma membrane; extracellular region; integral to membrane; actin filament; intercellular junction; lipid raft; cytoskeleton; membrane; perinuclear region of cytoplasm; apical plasma membrane; cytoplasm; basal plasma membrane; stress fiber; plasma membrane; cell junction
Molecular Function:transferase activity; protein binding; protein-tyrosine kinase activity; growth factor binding; transferase activity, transferring phosphorus-containing groups; nucleotide binding; kinase activity; receptor activity; transmembrane receptor protein tyrosine kinase activity; ATP binding; protein kinase activity
Biological Process: positive regulation of cell adhesion; peptidyl-tyrosine phosphorylation; heart development; protein amino acid autophosphorylation; cell-matrix adhesion; positive regulation of cytokine secretion during immune response; protein amino acid phosphorylation; regulation of cell migration; positive regulation of vascular endothelial growth factor receptor signaling pathway; cell-cell adhesion; positive regulation of focal adhesion formation; hemopoiesis; angiogenesis; vasculogenesis; Tie receptor signaling pathway; regulation of endothelial cell proliferation; response to retinoic acid; regulation of establishment and/or maintenance of cell polarity; positive regulation of phosphoinositide 3-kinase activity; regulation of angiogenesis; positive regulation of peptidyl-serine phosphorylation; protein oligomerization; positive regulation of phosphoinositide 3-kinase cascade; patterning of blood vessels; 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; endothelial cell proliferation; positive regulation of protein amino acid phosphorylation; sprouting angiogenesis; phosphorylation; transmembrane receptor protein tyrosine kinase signaling pathway; negative regulation of apoptosis
|NCBI GenInfo Identifier:||730947|
|NCBI Gene ID:||21687|
|UniProt Related Accession:||Q02858|
|Molecular Weight:||125,701 Da|
|NCBI Full Name:||Angiopoietin-1 receptor|
|NCBI Synonym Full Names:||endothelial-specific receptor tyrosine kinase|
|NCBI Official Symbol:||Tek|
|NCBI Official Synonym Symbols:||Hyk; STK1; Tie2; Tie-2; Cd202b; AA517024|
|NCBI Protein Information:||angiopoietin-1 receptor; p140 TEK; 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; HYK; STK1; 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; mTIE2; p140 TEK; CD_antigen: CD202b|
|Protein Family:||Angiopoietin-1 receptor|
|UniProt Gene Name:||Tek|
|UniProt Entry Name:||TIE2_MOUSE|