Western blot analysis of extracts from COS cells, untransfected or transfected with mouse angiopoietin-2, using Angiopoietin-2 Antibody.
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 datasheet or 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 November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised October 2016
Protocol Id: 10
Angiopoietin-2 Antibody detects transfected levels of mouse angiopoietin-2 protein.Species Reactivity:
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Lys275 of mouse angiopoietin-2. Antibodies are purified by peptide affinity chromatography.
Angiopoietins are a family of Tie receptor ligands. There are four angiopoietins discovered so far: angiopoietins 1, 2, 3 and 4 (Ang1, 2, 3, and 4) (1-3). Ang1 binds to the Tie-2 receptor and leads to its autophosphorylation and subsequent activation of downstream signaling pathways. It plays an important role in blood vessel formation, maturation and subsequent stabilization (1,4,5). Ang2 is an endothelium-specific growth factor that functions as an antagonist to Ang1, promotes vascular associated proinflammatory function, destabilizes quiescent endothelium, leads to vascular leakage and vascular destablization and remodeling (2,6,7). Ang2 is selectively expressed in many tumor tissues where, combined with other growth factors such as VEGF, it can promote vascular remodeling, angiogenesis and inflammation (7-9).
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