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13588
Phospho-p90RSK (Ser380) (D5D8) Rabbit mAb (Alexa Fluor® 488 Conjugate)
Antibody Conjugates

Phospho-p90RSK (Ser380) (D5D8) Rabbit mAb (Alexa Fluor® 488 Conjugate) #13588

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Flow cytometric analysis of Jurkat cells, untreated (blue) or treated with TPA #4174 (green), using Phospho-p90RSK (Ser380) (D5D8) Rabbit mAb (Alexa Fluor® 488 Conjugate).

To Purchase # 13588S
Product # Size Price
13588S
100 µl  (50 tests) $ 327

Supporting Data

REACTIVITY H M R Mk
SENSITIVITY Endogenous
MW (kDa)
Isotype Rabbit IgG

Application Key:

  • W-Western
  • IP-Immunoprecipitation
  • IHC-Immunohistochemistry
  • ChIP-Chromatin Immunoprecipitation
  • IF-Immunofluorescence
  • F-Flow Cytometry
  • E-P-ELISA-Peptide

Species Cross-Reactivity Key:

  • H-Human
  • M-Mouse
  • R-Rat
  • Hm-Hamster
  • Mk-Monkey
  • Mi-Mink
  • C-Chicken
  • Dm-D. melanogaster
  • X-Xenopus
  • Z-Zebrafish
  • B-Bovine
  • Dg-Dog
  • Pg-Pig
  • Sc-S. cerevisiae
  • Ce-C. elegans
  • Hr-Horse
  • All-All Species Expected

Product Description

This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 488 fluorescent dye and tested in-house for direct flow cytometry analysis in human and mouse cells. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated Phospho-p90RSK (Ser380) (D5D8) Rabbit mAb #12032.

Product Usage Information

Application Dilution
Flow Cytometry 1:50

Storage:

Supplied in PBS (pH 7.2), less than 0.1% sodium azide and 2 mg/ml BSA. Store at 4°C. Do not aliquot the antibody. Protect from light. Do not freeze.

Protocol

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Flow Cytometry, Methanol Permeabilization Protocol for Directly Conjugated Antibodies

A. Solutions and Reagents

All reagents required for this protocol may be efficiently purchased together in our Intracellular Flow Cytometry Kit (Methanol) #13593, or individually using the catalog numbers listed below.

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 1X Phosphate Buffered Saline (PBS): To prepare 1 L 1X PBS: add 100 ml 10X PBS (#12528) to 900 ml water mix.
  2. 4% Formaldehyde, Methanol-Free (#47746)
  3. 100% Methanol (#13604): Chill before use
  4. Antibody Dilution Buffer: Purchase ready-to-use Flow Cytometry Antibody Dilution Buffer (#13616), or prepare a 0.5% BSA PBS buffer by dissolving 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

NOTE: When including fluorescent cellular dyes in your experiment (including viability dyes, DNA dyes, etc.), please refer to the dye product page for the recommended protocol. Visit www.cellsignal.com/flowdyes for a listing of cellular dyes validated for use in flow cytometry.

B. Fixation

NOTE: Adherent cells or tissue should be dissociated and in single-cell suspension prior to fixation.

NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, 150-300g for 1-5 minutes will be sufficient to pellet the cells.

NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.

NOTE: Antibodies targeting CD markers or other extracellular proteins may be added prior to fixation if the epitope is disrupted by formaldehyde and/or methanol. The antibodies will remain bound to the target of interest during the fixation and permeabilization process. However, note that some fluorophores (including PE and APC) are damaged by methanol and thus should not be added prior to permeabilization. Conduct a small-scale experiment if you are unsure.

  1. Pellet cells by centrifugation and remove supernatant.
  2. Resuspend cells in approximately 100 µl 4% formaldehyde per 1 million cells. Mix well to dissociate pellet and prevent cross-linking of individual cells.
  3. Fix for 15 min at room temperature (20-25°C).
  4. Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 0.5-1 ml 1X PBS. Proceed to Permeabilization step.
    1. Alternatively, cells may be stored overnight at 4°C in 1X PBS.

C. Permeabilization

  1. Permeabilize cells by adding ice-cold 100% methanol slowly to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol.
  2. Permeabilize for a minimum of 10 min on ice.
  3. Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.

D. Immunostaining

NOTE: Count cells using a hemocytometer or alternative method.

  1. Aliquot desired number of cells into tubes or wells. (Generally, 5x105 to 1x106 cells per assay.)
  2. Wash cells by centrifugation in excess 1X PBS to remove methanol. Discard supernatant in appropriate waste container. Repeat if necessary.
  3. Resuspend cells in 100 µl of diluted primary antibody, prepared in Antibody Dilution Buffer at a recommended dilution or as determined via titration.
  4. Incubate for 1 hr at room temperature. Protect from light.
  5. Wash by centrifugation in Antibody Dilution Buffer or 1X PBS. Discard supernatant. Repeat.
  6. Resuspend cells in 200-500 µl of 1X PBS and analyze on flow cytometer.

posted July 2009

revised August 2019

Protocol Id: 407

Specificity / Sensitivity

Phospho-p90RSK (Ser380) (D5D8) Rabbit mAb (Alexa Fluor® 488 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

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser377 of human p90RSK3 protein.

Background

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).

  1. Fisher, T.L. and Blenis, J. (1996) Mol Cell Biol 16, 1212-9.
  2. Smith, J.A. et al. (1999) J Biol Chem 274, 2893-8.
  3. Dalby, K.N. et al. (1998) J Biol Chem 273, 1496-505.

Pathways & Proteins

Explore pathways + proteins related to this product.

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 conjugates in this product are sold under license from Life Technologies Corporation, for research use only excluding use in combination with DNA microarrays and high content screening (HCS).
This product is provided under an intellectual property license from Life Technologies Corporation. The transfer of this product is conditioned on the buyer using the purchased product solely in research conducted by the buyer, excluding contract research or any fee for service research, and the buyer must not (1) use this product or its components for (a) diagnostic, therapeutic or prophylactic purposes; (b) testing, analysis or screening services, or information in return for compensation on a per-test basis; or (c) manufacturing or quality assurance or quality control, and/or (2) sell or transfer this product or its components for resale, whether or not resold for use in research. For information on purchasing a license to this product for purposes other than as described above, contact Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, CA 92008 USA or outlicensing@lifetech.com.