Western blot analysis of extracts from mouse and rat heart tissue using Myosin Light Chain 2v (D5I1C) (Cardiac Isoform) Rabbit mAb (Rodent Specific).
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. 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
Myosin Light Chain 2v (D5I1C) (Cardiac Isoform) Rabbit mAb (Rodent Specific) recognizes endogenous levels of total myosin light chain 2v (cardiac isoform) protein.Species Reactivity:
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of mouse myosin light chain 2v (cardiac isoform) protein.
Myosin is composed of six polypeptide chains: two identical heavy chains and two pairs of light chains. Myosin light chain 2 (MLC2), also known as myosin regulatory light chain (MRLC), RLC, or LC20, has many isoforms with varying tissue distribution. Smooth muscle MLC2 is phosphorylated at Thr18 and Ser19 by myosin light chain kinase (MLCK) in a Ca2+/calmodulin-dependent manner (1). These phosphorylation events are correlated with myosin ATPase activity and smooth muscle contraction (2). Striated muscle contraction is regulated by the troponin-tropomyosin complex in thin actin filaments and by binding of Ca2+ to troponin C (3). Two types of myosin light chain are expressed in the heart, with myosin light chain 2v (MYL2, MLC-2v) expression restricted to the ventricles and myosin light chain 2a (MYL7, MLC-2a) found specific to the atria. Mutations in the corresponding MYL2 gene are found in patients diagnosed with a form of hypertrophic cardiomyopathy characterized by thickening of the mid-left ventricle.
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