|H M R Mk||Endogenous||48, 50||Rabbit|
Western blot analysis of extracts from NIH/3T3 and C6 cells, untreated, 8-Br-cGMP-treated, 8-Br-cAMP-treated or forskolin-treated as indicated, using Phospho-VASP (Ser239) Antibody.Learn more about how we get our images.
Western blot analysis of extracts from A-431 cells, untreated (-) or treated with Forskolin #3828 (+), using Phospho-VASP (Ser239) Antibody (upper) and VASP (9A2) Rabbit mAb #3132 (lower).Learn more about how we get our images.
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
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.
Phospho-VASP (Ser239) Antibody detects endogenous levels of VASP only when phosphorylated at serine 239. The antibody does not cross-react with phosphorylated VASP homologues, such as Mena.
Human, Mouse, Rat, Monkey
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser239 of human VASP. Antibodies are purified by protein A and peptide affinity chromatography.
Vasodilator-stimulated phosphoprotein (VASP) was originally characterized as a substrate of both cGMP- and cAMP-dependent kinases (PKG and PKA, or cGPK and cAPK, respectively) (1). It is now believed that VASP belongs to the Ena/VASP family of adaptor proteins linking the cytoskeletal system to the signal transduction pathways and that it functions in cytoskeletal organization, fibroblast migration, platelet activation and axon guidance (2,3). Three phosphorylation sites, Ser157, Ser239, and Thr278, have been identified. Ser239 is the major PKG phosphorylation site while Ser157 is the major PKA phosphorylation site (4). Evidence suggests that VASP phosphorylation reduces its association with actin and has a negative effect on actin polymerization (5). Phosphorylation at Ser239 of VASP is a useful marker for monitoring PKG activation and signaling (6,7).
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