Analysis of TIE2 (pY992) in human vascular endothelium. EA-hy 926 cells (Edgell, McDonald, Graham, 1983) were serum starved overnight, detached using 1X trypsin, washed, resuspended in serum-free DMEM and rested for 20 minutes at 37°C, and then either left unstimulated (open histogram) or stimulated with 1 mM Pervanadate (Sigma Cat. No. S6508, shaded histogram) for 20 minutes at 37°C (left figure). The cells were fixed (BD Cytofix™ buffer, Cat. No. 554655) for 10 minutes at 37°C, then permeabilized (BD Phosflow™ Perm Buffer III, Cat. No. 558050) on ice for at least 30 minutes, and then stained with Alexa Fluor® 647 Mouse anti-TIE2 (pY992, Cat. No. 560051). Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system. The specificity of mAb K93-754 was confirmed by western blot using unconjugated antibody, at 0.25, 0.125 and 0.063 μg/ml (lanes 1, 2, and 3, respectively) on lysates from control (left panel) and Pervanadate-treated (right panel) EA-hy 926 cells (right figure). TIE2 (pY1102) is identified as a band of 160 kDa in the treated cells.
Analysis of TIE2 (pY992) in human vascular endothelium. EA-hy 926 cells (Edgell, McDonald, Graham, 1983) were serum starved overnight, detached using 1X trypsin, washed, resuspended in serum-free DMEM and rested for 20 minutes at 37°C, and then either left unstimulated (open histogram) or stimulated with 1 mM Pervanadate (Sigma Cat. No. S6508, shaded histogram) for 20 minutes at 37°C (left figure). The cells were fixed (BD Cytofix™ buffer, Cat. No. 554655) for 10 minutes at 37°C, then permeabilized (BD Phosflow™ Perm Buffer III, Cat. No. 558050) on ice for at least 30 minutes, and then stained with Alexa Fluor® 647 Mouse anti-TIE2 (pY992, Cat. No. 560051). Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system. The specificity of mAb K93-754 was confirmed by western blot using unconjugated antibody, at 0.25, 0.125 and 0.063 μg/ml (lanes 1, 2, and 3, respectively) on lysates from control (left panel) and Pervanadate-treated (right panel) EA-hy 926 cells (right figure). TIE2 (pY1102) is identified as a band of 160 kDa in the treated cells.
Product Details
BD Phosflow™
TEK, VMCM, CD202b
Human (QC Testing), Mouse (Predicted)
Mouse BALB/c IgG1, κ
Phosphorylated Human TIE2 Peptide
Intracellular staining (flow cytometry) (Routinely Tested)
20 µl
AB_1645447
Aqueous buffered solution containing BSA and ≤0.09% sodium azide.
RUO
Preparation And Storage
Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated to Alexa Fluor® 647 under optimum conditions, and unreacted Alexa Fluor® 647 was removed.
Recommended Assay Procedures
Either BD Cytofix™ fixation buffer or BD Phosflow™ Fix Buffer I may be used for cell fixation.
Product Notices
- This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
- Alexa Fluor® 647 fluorochrome emission is collected at the same instrument settings as for allophycocyanin (APC).
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- The Alexa Fluor®, Pacific Blue™, and Cascade Blue® dye antibody conjugates in this product are sold under license from Molecular Probes, Inc. for research use only, excluding use in combination with microarrays, or as analyte specific reagents. The Alexa Fluor® dyes (except for Alexa Fluor® 430), Pacific Blue™ dye, and Cascade Blue® dye are covered by pending and issued patents.
- Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
Companion Products
Fixation Buffer RUO
Size
100 mL
Cat No.
554655
Fix Buffer I RUO
Size
250 mL
Cat No.
557870
Perm Buffer III RUO
Size
125 mL
Cat No.
558050
560051 Rev. 1
Antibody Details
K91-860
TIE2 (Tyrosine kinase with Immunoglobulin-like and EGF-like domains 2), also known as TEK (Tunical Endothelial Kinase), is an endothelial and hematopoietic cell-specific receptor tyrosine kinase (RTK) that is critical to the development and maintenance of the vasculature and highly conserved among vertebrate species. The angiopoietins are ligands of TIE2, and the abnormal vascular growth that often occurs in solid tumors is a result of disruptions in the coordinated actions of the angiopoietins, TIE2, and the closely related TIE1 RTK. Upon activation by angiopoietins, TIE2 autophosphorylates at least 2 tyrosines in its protein kinase domain and at least 3 tyrosines in its C-terminal tail.
The K91-860 monoclonal antibody recognizes the phosphorylated tyrosine 992 (Y992) in the protein kinase domain of activated TIE2. The orthologous phosphorylation site in mouse TIE2 is Y990.
560051 Rev. 1
Format Details
Alexa Fluor™ 647
Alexa Fluor™ 647 Dye is part of the BD red family of dyes. This is a small organic fluorochrome with an excitation maximum (Ex Max) at 653-nm and an emission maximum (Em Max) at 669-nm. Alexa Fluor 647 is designed to be excited by the Red laser (627-640 nm) and detected using an optical filter centered near 520-nm (e.g., a 660/20 nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
Alexa Fluor™ 647
Red 627-640 nm
653 nm
669 nm
560051 Rev.1
Citations & References
Development References (6)
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Audero E, Cascone I, Maniero F, et al. Adaptor ShcA protein binds tyrosine kinase Tie2 receptor and regulates migration and sprouting but not survival of endothelial cells. J Biol Chem. 2004; 279(13):13224-13233. (Biology).
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DeBusk LM, Hallahan DE, Lin PC. Akt is a major angiogenic mediator downstream of the Ang1/Tie2 signaling pathway. Exp Cell Res. 2004; 298(1):167-177. (Biology).
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Edgell C-JS, McDonald CC, Graham JB. Permanent cell line expressing human factor VIII-related antigen established by hybridization. Proc Natl Acad Sci U S A. 1983; 80:3734-3737. (Methodology: Controls).
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Jones N, Iljin K, Dumont DJ, Alitalo K. Tie receptors: new modulators of angiogenic and lymphangiogenic responses. Mol Cell Biol. 2001; 2:257-267. (Biology).
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Murray BW, Padrique ES, Pinko C, McTigue MA. Mechanistic effects of autophosphorylation on receptor tyrosine kinase catalysis: enzymatic characterization of Tie2 and phospho-Tie2.. Biochemistry. 2001; 40(34):10243-10253. (Biology).
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Peters KG, Kontos CD, Lin PC, et al. Functional significance of Tie2 signaling in the adult vasculature. Recent Prog Horm Res. 2004; 59:51-71. (Biology).
560051 Rev. 1
Please refer to Support Documents for Quality Certificates
Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described
Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims. Comparisons are not made against non-BD technologies, unless otherwise noted.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
