Western blot analysis of purified RSK1, RSK2 and RSK3 proteins using RSK1 Antibody #9333 (upper), RSK2 Antibody #9340 (middle) or RSK3 Antibody (lower).
Western blot analysis of extracts from COS cells, untransfected or transfected with RSK3, using RSK3 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
RSK3 Antibody detects transfected levels of RSK3 protein. It does not cross-react with the RSK1 or RSK2 isoforms.Species Reactivity:
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro709 of human RSK3. 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).
Unlike other RSK isoforms, the N-terminal region of RSK3 contains a putative nuclear localization signal and the kinase translocates to the nucleus in serum-starved HeLa cells (4). RSK3 is expressed ubiquitously in human tissues and localizes to developing neural and sensory areas in the mouse (5). Altered expression and mutations in the corresponding gene are associated with breast cancer and in some cancer cell lines, suggesting a possible role for this kinase as a tumor suppressor (6).
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