Western blot analysis of extracts from HeLa, COS and NIH/3T3 cells, treated with lambda-phosphatase or TPA/UV, using Phospho-RSK2 (Ser227) (D53A11) Rabbit mAb.
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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 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 June 2020
Protocol Id: 10
Phospho-RSK2 (Ser227) (D53A11) Rabbit mAb detects endogenous levels of RSK2 only when phosphorylated at Ser227. It shows cross-reactivity with RSK1 when phosphorylated at the homologous serine residues.
Human, Mouse, Rat, Monkey
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser227 of human RSK2.
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).
PDK1 phosphorylates Ser227 in the activation loop of the amino-terminal kinase domain of RSK2, leading to substantial activation of the kinase in vitro and in vivo. The constitutively active PDK1 cooperates with ERK in the activation of RSK following the exposure of cells to growth factors or UV-light (4, 5).
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