Western blot analysis of extracts from HeLa and C2C12 cells, untreated or nocodazole-treated, using Phospho-Stathmin (Ser16) Antibody.
|REACTIVITY||H M R Hm Mk|
|MW (kDa)||19, 20|
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
Phospho-Stathmin (Ser16) Antibody recognizes endogenous levels of stathmin protein only when phosphorylated at Ser16.Species Reactivity:
Human, Mouse, Rat, Hamster, Monkey
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser16 of human stathmin. Antibodies are purified using protein A and peptide affinity chromatography.
Stathmin is a ubiquitously expressed microtubule destabilizing phosphoprotein that is upregulated in a number of cancers. The amino terminus of the protein contains multiple phosphorylation sites and is involved in the promotion of tubulin filament depolymerization. Phosphorylation at these sites inactivates the protein and stabilizes microtubules. Ser16 phosphorylation by CaM kinases II and IV (1,2) increases during G2/M-phase and is involved in mitotic spindle regulation (3,4). Ser38 is a target for cdc2 kinase (5) and TNF-induced cell death gives rise to reactive oxygen intermediates leading to hyperphosphorylation of stathmin (6). EGF receptor activation of Rac and cdc42 also increases phosphorylation of stathmin on Ser16 and Ser38 (7). Other closely related family members are neuronally expressed and include SCG10, SCLIP, RB3 and its splice variants RB3' and RB3''. Stathmin and SCG10 have been shown to play roles in neuronal-like development in PC-12 cells (8).
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