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      PE Mouse anti-Stat1 (pS727)
      PE Mouse anti-Stat1 (pS727)
      Analysis of Stat1 (pS727) in human epithelioid carcinoma and peripheral blood lymphocytes.  HeLa S3 cells (ATCC CCL 2.2, left panel) were either treated with 0.5 μg/ml Nocodazole (Sigma-Aldrich, Cat. No. M1404) at 37°C for 18 hours (dashed-line histogram) or untreated (solid-line histogram).  The cells were fixed (BD Cytofix™ buffer, Cat. No. 554655) for 10 minutes at 37°C, then permeabilized (BD Phosflow™ Perm Buffer III, Cat. No. 558050) on ice for at least 30 minutes, and then stained with PE Mouse anti-Stat1 (pS727, Cat. No. 560069).  Human peripheral blood mononuclear cells (PBMC, right panel) were either stimulated with 50 nM PMA (Sigma, P8139) for 15 minutes at 37ºC (shaded histogram) or untreated (open histogram).  The PBMC were fixed (BD Cytofix™ buffer, Cat. No. 554655) for 10 minutes at 37°C, permeabilized with BD Phosflow™ Perm Buffer III (Cat. No. 558050) on ice for 30 minutes, and then stained with PE Mouse anti-Stat1 (pS727).  For data analysis, lymphocytes were selected by their scatter profile.  All flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.
      PE Mouse anti-Stat1 (pS727)
      The specificity of mAb K51-856 was confirmed by western blot. Lysates from PMA-treated cells (lane 1, left blot), control PBMC (lane 2, left blot), Nocadazole-treated cells (lane 1, right blot), and control HeLa S3 cells (lane 2, right blot) were treated with unconjugated antibody at 2.0 ug/ml.  Stat1 (pS727) is identified as a band of 91 kDa in the treated cells.
      PE Mouse anti-Stat1 (pS727) PE Mouse anti-Stat1 (pS727) PE Mouse anti-Stat1 (pS727) PE Mouse anti-Stat1 (pS727)
      Analysis of Stat1 (pS727) in human epithelioid carcinoma and peripheral blood lymphocytes.  HeLa S3 cells (ATCC CCL 2.2, left panel) were either treated with 0.5 μg/ml Nocodazole (Sigma-Aldrich, Cat. No. M1404) at 37°C for 18 hours (dashed-line histogram) or untreated (solid-line histogram).  The cells were fixed (BD Cytofix™ buffer, Cat. No. 554655) for 10 minutes at 37°C, then permeabilized (BD Phosflow™ Perm Buffer III, Cat. No. 558050) on ice for at least 30 minutes, and then stained with PE Mouse anti-Stat1 (pS727, Cat. No. 560069).  Human peripheral blood mononuclear cells (PBMC, right panel) were either stimulated with 50 nM PMA (Sigma, P8139) for 15 minutes at 37ºC (shaded histogram) or untreated (open histogram).  The PBMC were fixed (BD Cytofix™ buffer, Cat. No. 554655) for 10 minutes at 37°C, permeabilized with BD Phosflow™ Perm Buffer III (Cat. No. 558050) on ice for 30 minutes, and then stained with PE Mouse anti-Stat1 (pS727).  For data analysis, lymphocytes were selected by their scatter profile.  All flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.
      The specificity of mAb K51-856 was confirmed by western blot. Lysates from PMA-treated cells (lane 1, left blot), control PBMC (lane 2, left blot), Nocadazole-treated cells (lane 1, right blot), and control HeLa S3 cells (lane 2, right blot) were treated with unconjugated antibody at 2.0 ug/ml.  Stat1 (pS727) is identified as a band of 91 kDa in the treated cells.
      Product Details
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      BD Phosflow™
      Human (QC Testing), Mouse (Predicted)
      Mouse BALB/c IgG1, κ
      Phosphorylated Human Stat1 Peptide
      Intracellular staining (flow cytometry) (Routinely Tested)
      20 µl
      AB_1645534
      Aqueous buffered solution containing BSA and ≤0.09% sodium azide.
      RUO


      Preparation And Storage

      Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated with R-PE under optimum conditions, and unconjugated antibody and free PE were removed.
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      Recommended Assay Procedures

      Either BD Cytofix™ fixation buffer or BD Phosflow™ Fix Buffer I may be used for cell fixation.  Any of the three BD Phosflow™ permeabilization buffers may be used.

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      Product Notices

      1. This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
      2. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
      3. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
      4. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
      5. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
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      560069 Rev. 3
      Antibody Details
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      K51-856

      Stat (Signal transducer and activators of transcription) proteins are critical mediators of the biologic activity of cytokines, including interleukins, interferons, erythropoietin, and growth factors.  Ligand-receptor interaction leads to activation of constitutively associated JAK family kinases and subsequent recruitment/activation of Stat proteins by tyrosine phosphorylation.  Active Stat proteins then move to the nucleus to promote transcription of cytokine-inducible genes.  Seven Stat proteins have been cloned, each of which is differentially expressed and/or activated in a cytokine-specific and cell type-specific manner.  Stat1 and Stat2 are components of the ISGF3 (Interferon-Stimulated Gene Factor 3) complex, which is the primary transcription activator induced by the binding of the interferon to a specific cell-surface receptor.  Stat1 has two alternatively spliced isoforms, 91-kDa Stat1α and 84-kDa Stat1β; Stat1α has 38 additional C-terminal amino acids.  In response to the binding of IFNα, IFNγ, EGF, PDGF, or CSF-1 to their respective receptors, the Stat1 subunits become tyrosine-phosphorylated at Y701, and the complex is translocated to the nucleus. This results in the formation of an active complex that includes the DNA-binding p48 subunit.  This complex is responsible for modulating the transcription of the interferon-stimulated genes (ISGs).  Furthermore, phosphorylation of serine 727 (S727) of Stat1 may occur in response to pathogens, cytokines, UV irradiation, and engagement of T and B cell antigen receptors.  This second phosphorylation event is necessary for fully effective activation of transcription and may differentially regulate gene activation by interacting with other regulatory factors, such as p53 in the apoptotic pathway.

      The K51-856 monoclonal antibody recognizes the phosphorylated S727 in the C-terminal transactivating domain of human Stat1α; this site is not present in Stat1β.

      560069 Rev. 3
      Format Details
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      PE
      R-Phycoerythrin (PE), is part of the BD family of Phycobiliprotein dyes. This fluorochrome is a multimeric fluorescent phycobiliprotein with excitation maximum (Ex Max) of 496 nm and 566 nm and an emission maximum (Em Max) at 576 nm. PE is designed to be excited by the Blue (488 nm), Green (532 nm) and Yellow-Green (561 nm) lasers and detected using an optical filter centered near 575 nm (e.g., a 575/26-nm bandpass filter). As PE is excited by multiple lasers, this can result in cross-laser excitation and fluorescence spillover on instruments with various combinations of Blue, Green, and Yellow-Green lasers. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
      altImg
      PE
      Yellow-Green 488 nm, 532 nm, 561 nm
      496 nm, 566 nm
      576 nm
      560069 Rev.3
      Citations & References
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      View product citations for antibody "560069" on CiteAb

      Development References (7)

      1. Bromberg J, Darnell JE. The role of STATs in transcriptional control and their impact on cellular function. Oncogene. 2000; 19(21):2468-2473. (Biology). View Reference
      2. Gamero AM, Larner AC. Signaling via the T cell antigen receptor induces phosphorylation of Stat1 on serine 727. J Biol Chem. 2000; 275(22):16574-16578. (Biology).
      3. Heim MH. The Jak-STAT pathway: specific signal transduction from the cell membrane to the nucleus. Eur J Clin Invest. 1996; 26(1):1-12. (Biology). View Reference
      4. Kovarik P, Mangold M, Ramsauer K, et al. Specificity of signaling by STAT1 depends on SH2 and C-terminal domains that regulate Ser727 phosphorylation, differentially affecting specific target gene expression. EMBO J. 2001; 20(1 & 2):91-100. (Biology).
      5. Nguyen H, Ramana CV, Bayes J, Stark GR. Roles of phosphatidylinositol 3-kinase in interferon-γ-dependent phosphorylation of STAT1 on serine 727 and activation of gene expression. J Biol Chem. 2001; 276(36):33361-33368. (Biology).
      6. Townsend PA, Scarabelli TM, Davidson SM, Knight RA, Latchman DS, Stephanou A. STAT-1 interacts with p53 to enhance DNA damage-induced apoptosis. J Biol Chem. 2004; 279(7):5811-5820. (Biology).
      7. Varinou L, Ramsauer K, Karaghiosoff M, et al. Phosphorylation of the Stat1 transactivation domain is required for full-fledged IFN-γ-dependent innate immunity. Immunity. 2003; 19:793-802. (Biology).
      View All (7) View Less
      560069 Rev. 3

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      Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described

       

      Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims.  Comparisons are not made against non-BD technologies, unless otherwise noted.

      For Research Use Only. Not for use in diagnostic or therapeutic procedures.