Western blot analysis of extracts from Sf9 cells overexpressing GST-human Tie2 kinase domain fusion proteins, wild-type (lane 1) S1119D mutant (lane 2), S1119A mutant (lane 3) or kinase-dead (lane 4) , using Phospho-Tie2 (Ser1119) Antibody (upper) or GST antibody (lower). The wild-type Tie2 kinase domain is constitutively phosphorylated when overexpressed in Sf9 cells. The putative molecular weight of GST-Tie2 fusion is approximately 65 kDa.
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody product webpage for recommended antibody dilution.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised June 2020
Protocol Id: 10
Phospho-Tie2 (Ser1119) Antibody detects transfected levels of Tie2 protein only when phosphorylated at serine 1119.
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser1119 of human Tie2. Antibodies are purified by protein A and peptide affinity chromatography.
Tie2/Tek is a receptor tyrosine kinase (RTK) expressed almost exclusively on endothelial cells. It is critical for vasculogenesis and could be important for maintaining endothelial cell survival and integrity in adult blood vessels as well as tumor angiogenesis (1-3). A family of ligands known as the angiopoietins binds to Tie2. Interestingly, these ligands appear to have opposing actions; Angiopoietin-1 (Ang1) and Angiopoietin-4 (Ang4) stimulate tyrosine phosphorylation of Tie2; Angiopoietin-2 (Ang2) and Angiopoietin-3 (Ang3) can inhibit this phosphorylation (4,5). Downstream signaling components, including Grb2, Grb7, Grb14, SHP-2, the p85 subunit of phosphatidylinositol 3-kinase, and p56/Dok-2 interact with Tie2 in a phosphotyrosine-dependent manner through their SH2 or PTB domains (6,7). Tyr992 is located on the putative activation loop of Tie2 and is a major autophosphorylation site (8).
Phosphorylation of Ser1119 at the carboxy-terminus of Tie2 results in an increase of Tie2 kinase activity (personal communication with Dr. Chris Kontos, Duke University).
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