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Phospho-p90RSK (Ser380) (D5D8) Rabbit mAb (Alexa Fluor® 647 Conjugate) #13575
Flow cytometric analysis of Jurkat cells, U0126 (10uM, 2hr; blue) or treated with TPA (200uM, 30min; green) using Phospho-p90RSK (Ser380) (D5D8) Rabbit mAb (Alexa Fluor® 647 Conjugate) (solid lines) or concentration-matched
Rabbit (DA1E) mAb IgG XP® Isotype Control (Alexa Fluor® 647 Conjugate) #2985 (dashed lines).Learn more about how we get our images
Gallery: Phospho-p90RSK (Ser380) (D5D8) Rabbit mAb (Alexa Fluor® 647 Conjugate) #13575
Flow Cytometry, Methanol Permeabilization Protocol for Direct Conjugates
A. Solutions and Reagents
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
- 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
- 16% Formaldehyde (methanol free).
- 100% methanol.
- Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.
NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.
- Collect cells by centrifugation and aspirate supernatant.
- Resuspend cells in 0.5-1 ml 1X PBS. Add formaldehyde to obtain a final concentration of 4%.
- Fix for 15 min at room temperature.
- Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 0.5-1 ml 1X PBS.
- Permeabilize cells by adding ice-cold 100% methanol slowly to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol.
- Incubate 30 min on ice.
- Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.
- Aliquot desired number of cells into tubes or wells.
- Wash cells by centrifugation in excess 1X PBS to remove methanol. Discard supernatant in appropriate waste container. Repeat if necessary.
- Resuspend cells in 100 µl of diluted antibody conjugate (prepared in incubation buffer at the recommended dilution).
- Incubate for 1 hr at room temperature. Protect from light.
- Wash by centrifugation in incubation buffer. Discard supernatant. Repeat.
- Resuspend cells in 1X PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to optional DNA stain (Section E).
E. Optional DNA Dye
- Resuspend cells in 0.5 ml of DNA dye (e.g. Propidium Iodide (PI)/RNase Staining Solution #4087).
- Incubate for at least 5 min at room temperature.
- Analyze cells in DNA staining solution on flow cytometer.
posted July 2009
revised June 2017
Phospho-p90RSK (Ser380) (D5D8) Rabbit mAb (Alexa Fluor® 647 Conjugate) recognizes endogenous levels of RSK1, RSK2, and RSK3 proteins only when phosphorylated at Ser380 (RSK1), Ser386 (RSK2), or Ser377 (RSK3).Species Reactivity: Human, Mouse, Rat, Monkey Species predicted to react based on 100% sequence homology: Chicken, Xenopus, Zebrafish, Bovine, Dog, Pig, Horse
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser377 of human p90RSK3 protein.
This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 647 fluorescent dye and tested in-house for direct flow cytometry analysis in human cells. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated Phospho-p90RSK (Ser380) (D5D8) Rabbit mAb #12032.
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).
Upon mitogenic stimulation, p44/42 ERK1/2 and ERK5 MAP kinases cooperatively phosphorylate p90RSK at Thr573 (p90RSK1 numbering) located within the carboxy-terminal kinase domain and at Thr359/Ser363 in the linker region between the two kinase domains (3). Phosphorylation of p90RSK at Thr573 within the activation loop of the p90RSK carboxy-terminal kinase domain promotes activation and directs phosphorylation of Ser380 within the hydrophobic stretch of the linker region (4,5). The p90RSK phosphorylated at Ser380 acts as a docking site for the constitutively active Ser/Thr kinase PDK1, which in turn phosphorylates Ser221 within the amino-terminal kinase domain activation loop, resulting in full enzymatic activation of the p90RSK (6). Antibodies against these phosphorylation sites are useful for understanding the kinetics and regulation of p90RSK activation.
For more information regarding the phospho-regulatory sites within each RSK isoform, including more information regarding the seminal studies demonstrating the complex phosphorylation cascades involved, please see the references herein and PhosphoSitePlus® (www.phosphosite.org).
For Research Use Only. Not For Use In Diagnostic Procedures. Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. Alexa Fluor is a registered trademark of Life Technologies Corporation. The Alexa Fluor dye antibody conjugates in this product are sold under license from Life Technologies Corporation for research use only, except for use in combination with DNA microarrays. The Alexa Fluor® dyes (except for Alexa Fluor® 430 dye) are covered by pending and issued patents. Alexa Fluor® is a registered trademark of Molecular Probes, Inc.