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
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.
M-RIP (D9I5T) Rabbit mAb recognizes endogenous levels of total M-RIP protein.
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro165 of human M-RIP protein.
Myosin phosphatase-rho interacting protein (M-RIP), also known as p116RIP, RIP3, and MPRIP, localizes to actin-myosin filaments regulating cytoskeletal dynamics (1-3). M-RIP contains amino-terminal pleckstrin homology domains, carboxyl-terminal coiled-coil domains, and was originally identified to associate with the myosin phosphatase complex. M-RIP binds to MBS/MYRT, the myosin binding subunit of myosin phosphatase, as well as RhoA (1-3). Phosphorylation of MYRT by Rho-associated kinase (ROCK) inhibits myosin phosphatase activity, resulting in increased levels of phosphorylation on myosin light chain, and enhanced contractility (4,5). M-RIP may function as a scaffolding protein for the complex between the myosin phosphatase complex, Rho/ROCK, and actin (2,6). Silencing of M-RIP results in disassembly of the complex, increased phosphorylation of myosin light chain, and changes to cytoskeletal dynamics (7,8).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. Tween is a registered trademark of ICI Americas, Inc.
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|14383S||100 µl (10 western blots)||$ 255.0|