Western blot analysis of extracts from HeLa, A-431 and 293T cells, untreated or treated with λ phosphatase and calf intestinal phosphatase (CIP), using Phospho-Myosin IIa (Ser1943) Antibody (upper) or Myosin IIa Antibody #3403 (lower).
Western blot analysis of extracts from A-431 cells, untreated or treated with λ phosphatase and calf intestinal phosphatase (CIP), using Phospho-Myosin IIa (Ser1943) Antibody. The antibody was pre-incubated with peptides corresponding to unphosphorylated Myosin IIa or Myosin IIa phosphorylated at Ser1943, as indicated.
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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-Myosin IIa (Ser1943) Antibody detects endogenous levels of myosin IIa protein only when phosphorylated at Ser1943.Species Reactivity:
Human, Mouse, Rat, Monkey
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser1943 of human myosin IIa protein. Antibodies are purified using protein A and peptide affinity chromatography.
Nonmuscle myosin is an actin-based motor protein essential to cell motility, cell division, migration, adhesion, and polarity. The holoenzyme consists of two identical heavy chains and two sets of light chains. The light chains (MLCs) regulate myosin II activity and stability. The heavy chains (NMHCs) are encoded by three genes, MYH9, MYH10, and MYH14, which generate three different nonmuscle myosin II isoforms, IIa, IIb, and IIc, respectively (reviewed in 1). While all three isoforms perform the same enzymatic tasks, binding to and contracting actin filaments coupled to ATP hydrolysis, their cellular functions do not appear to be redundant and they have different subcellular distributions (2-5). The carboxy-terminal tail domain of myosin II is important in isoform-specific subcellular localization (6). Research studies have shown that phosphorylation of myosin IIa at Ser1943 contributes to the regulation of breast cancer cell migration (7).
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