Western blot analysis of extracts from various cell lines using Myosin VI (D5Y5A) Rabbit mAb.
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 VI (D5Y5A) Rabbit mAb recognizes endogenous levels of total myosin VI protein.Species Reactivity:
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Arg1131 of human myosin VI protein.
The myosin family of motor proteins drive ATP-dependent actin-based motility in eukaryotic cells and contain a conserved amino-terminal motor domain (reviewed in 1,2).
Myosin VI is an unconventional minus-end-directed myosin involved in the transport of vesicles and organelles within the cell, endocytosis, and organelle biogenesis (3-6). The movement of myosin VI and its cargo along actin filaments is unique among myosin family members in its mechanism; its tail domain structure allows it to take larger than predicted steps along the actin filament (reviewed in 1,7).
Myosin VI has been shown to regulate the polarized delivery of proteins to specialized subcellular locations, including the delivery of EGFR to the leading edge of migrating cells (8), as well as the delivery of specialized axonal proteins in neurons (9). Myosin VI has also been shown to mediate activity of the tumor suppressor p53 during DNA damage (10,11).
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