Western blot analysis of extracts from HeLa cells, untreated or H2O2-treated, using Phospho-Cofilin (Ser3) Antibody (A and B) or Cofilin Antibody #3312 (C and D). Membranes (B and D) were treated with alkaline phosphatase (CIP) to demonstrate the phospho-specificity the antibody.
Western blot analysis of extracts from asynchronous or mitotic C6, CHO and COS cells, using Phospho-Cofilin (Ser3) Antibody (upper) or Cofilin Antibody #3312 (lower).
|REACTIVITY||H M R Hm Mk B|
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-Cofilin (Ser3) Antibody detects endogenous levels of cofilin only when phosphorylated at serine 3. The antibody may cross-react with phosphorylated cofilin 2, the muscle isoform.Species Reactivity:
Human, Mouse, Rat, Hamster, Monkey, BovineSpecies predicted to react based on 100% sequence homology:
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser3 of human cofilin. Antibodies are purified by protein A and peptide affinity chromatography.
Cofilin and actin-depolymerization factor (ADF) are members of a family of essential conserved small actin-binding proteins that play pivotal roles in cytokinesis, endocytosis, embryonic development, stress response, and tissue regeneration (1). In response to stimuli, cofilin promotes the regeneration of actin filaments by severing preexisting filaments (2). The severing activity of cofilin is inhibited by LIMK or TESK phosphorylation at Ser3 of cofilin (3-5). Phosphorylation at Ser3 also regulates cofilin translocation from the nucleus to the cytoplasm (6).
Explore pathways + proteins related to this product.
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