Western blot analysis of purified RSK1, RSK2, and RSK3 proteins using RSK1 Antibody (upper), RSK2 Antibody #9340 (middle) or RSK3 Antibody #9343 (lower).
Western blot analysis of extracts from HeLa and COS cells using RSK1 Antibody.
|REACTIVITY||H M R Mk|
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
RSK1 Antibody detects endogenous levels of RSK1. It does not cross-react with the RSK2 or RSK3 isoforms.Species Reactivity:
Human, Mouse, Rat, MonkeySpecies predicted to react based on 100% sequence homology:
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Glu718 of human RSK1. Antibodies are purified by protein A and peptide affinity chromatography.
The 90 kDa ribosomal S6 kinases (RSK1-4) are a family of widely expressed Ser/Thr kinases characterized by two nonidentical, functional kinase domains (1) and a carboxy-terminal docking site for extracellular signal-regulated kinases (ERKs) (2). Several sites both within and outside of the RSK kinase domain, including Ser380, Thr359, Ser363, and Thr573, are important for kinase activation (3). RSK1-3 are activated via coordinated phosphorylation by MAPKs, autophosphorylation, and phosphoinositide-3-OH kinase (PI3K) in response to many growth factors, polypeptide hormones, and neurotransmitters (3).
PI3K-induced activation of RSK1 is mediated by the Ser/Thr kinase mTOR (mammalian target of rapamycin) (4,5). This activation of RSK1 selectively increases the translation of mRNA transcripts containing a tract of pyrimidine (TOP) motif. An association between RSK1 and specific PKA subunits depends upon RSK1 activation state and determines both intracellular localization and specific activity of the kinase (6). Evidence from animal models suggests that RSK1 is a key regulator of glucose homeostasis and cell size (7).
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