Western blot analysis of extracts from 293 cells transfected with MSK1 or various mutants of MSK1, then treated with TPA (200 nM) and analyzed using Phospho-MSK1 (Ser360) Antibody.
Western blot analysis of extracts from RSK1, RSK2, RSK3 or MSK1 transfected 293 cells, untreated or TPA-treated (200 nM), using Phospho-MSK1 (Ser360) Antibody.
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-MSK1 (Ser360) Antibody detects transfected MSK1 only when phosphorylated at Ser360. This antibody does not recognize MSK1 phosphorylated at other sites, nor does it cross-react with phosphorylated RSK1, RSK2 or RSK3.Species Reactivity:
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser360 of human MSK1. Antibodies are purified by protein A and peptide affinity chromatography.
MSK1, a mitogen and stress activated protein kinase, is activated by Erk as well as p38 MAPK in response to growth factors and cellular stress, respectively (1). MSK1 resembles RSK because it has two kinase domains connected by a regulatory linker region (2). Phosphorylation of RSK1 at Ser364 and Ser381 is critical for RSK1 activity (3). These sites are analogous to Ser360 and Ser376 of MSK1, which may be important for MSK1 activity as well.
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