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68826
Phospho-Stat2 (Tyr690) (D3P2P) Rabbit mAb (Alexa Fluor® 488 Conjugate)

Phospho-Stat2 (Tyr690) (D3P2P) Rabbit mAb (Alexa Fluor® 488 Conjugate) #68826

APPLICATIONS

REACTIVITY SENSITIVITY MW (kDa) Isotype
H R Endogenous Rabbit IgG
Flow Cytometry

Flow cytometric analysis of U266 cells, untreated (blue) or treated with IFN-alpha (10 ng/ml, 15 min; green), using Phospho-Stat2 (Tyr690) (D3P2P) Rabbit mAb (Alexa Fluor® 488 Conjugate) (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (Alexa Fluor® 488 Conjugate) #2975 (dashed lines).

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Flow Cytometry, Methanol Permeabilization Protocol for Direct Conjugates

A. Solutions and Reagents

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

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 16% Formaldehyde (methanol free).
  3. 100% methanol.
  4. Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

B. Fixation

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

  1. Collect cells by centrifugation and aspirate supernatant.
  2. Resuspend cells in 0.5-1 ml 1X PBS. Add formaldehyde to obtain a final concentration of 4%.
  3. Fix for 15 min at room temperature.
  4. Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 0.5-1 ml 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. Incubate 30 min on ice.
  3. Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.

D. Immunostaining

  1. Aliquot desired number of cells into tubes or wells.
  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 antibody conjugate (prepared in incubation buffer at the recommended dilution).
  4. Incubate for 1 hr at room temperature. Protect from light.
  5. Wash by centrifugation in incubation buffer. Discard supernatant. Repeat.
  6. 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

  1. Resuspend cells in 0.5 ml of DNA dye (e.g. Propidium Iodide (PI)/RNase Staining Solution #4087).
  2. Incubate for at least 5 min at room temperature.
  3. Analyze cells in DNA staining solution on flow cytometer.

posted July 2009

revised June 2017

Protocol Id: 407

Application Dilutions
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.

Phospho-Stat2 (Tyr690) (D3P2P) Rabbit mAb (Alexa Fluor® 488 Conjugate) recognizes endogenous levels of Stat2 protein only when phosphorylated at Tyr690.

Species Reactivity:

Human, Rat

Monoclonal antibody is produced by immunizing animals with a synthetic phospho-peptide corresponding to residues surrounding Tyr690 of human Stat2 protein.

This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 488 fluorescent dye and tested in-house for direct flow cytometric analysis in human cells. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated Phospho-Stat2 (Tyr690) (D3P2P) Rabbit mAb #88410.

Stat2 (113-kDa), originally purified from the nuclei of alpha-interferon-treated cells, is critical to the transcriptional responses induced by type I interferons, IFN-alpha/beta (1,2). Knockout mice with a targeted disruption of Stat2 have higher susceptibility to viral infection and altered responses to type I interferons (3). Stat2 is rapidly activated by phosphorylation at Tyr690 in response to stimulation by IFN-alpha/beta via associations with receptor-bound Jak kinases (4). Unlike other Stat proteins, Stat2 does not form homodimers. Instead, activated Stat2 forms a heterodimer with Stat1 and translocates to the nucleus. There, it associates with the DNA-binding protein p48 and forms the transcriptional activator complex, interferon-stimulated gene factor 3 (ISGF3), promoting transcription from the ISRE (5).

  1. Fu, X.Y. et al. (1992) Proc Natl Acad Sci U S A 89, 7840-3.
  2. Ihle, J.N. (2001) Curr Opin Cell Biol 13, 211-7.
  3. Park, C. et al. (2000) Immunity 13, 795-804.
  4. Improta, T. et al. (1994) Proc Natl Acad Sci U S A 91, 4776-80.
  5. Horvath, C.M. et al. (1996) Mol Cell Biol 16, 6957-64.
Entrez-Gene Id
6773
Swiss-Prot Acc.
P52630
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
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.

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68826S
100 µl (96 assays) $ 320.0