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Alexa Fluor® 647 Mouse anti-Stat1 (pS727)
Alexa Fluor® 647 Mouse anti-Stat1 (pS727)

Analysis of Stat1 (pS727) in human epithelioid carcinoma and peripheral blood lymphocytes.  HeLa S3 cells (left panel, ATCC CCL 2.2) were either treated with 0.5 μg/ml Nocodazole (Sigma-Aldrich, Cat. No. M1404) at 37°C for 18 hours (dashed line) or untreated (solid line).  Human peripheral blood mononuclear cells (middle panel) were either stimulated with 50 nM PMA (Sigma, P8139) for 15 minutes at 37ºC (shaded histogram) or untreated (open histogram).  The cells were fixed (BD Cytofix™ buffer, Cat. No. 554655) for 10 minutes at 37°C, permeabilized (BD Phosflow™ Perm Buffer III, Cat. No. 558050) on ice for 30 minutes, and then stained with Alexa Fluor® 647 Mouse anti-Stat1 (pS727, Cat. No. 560190).  Flow cytometry was performed on a BD™ LSR II (left) or FACSCalibur (middle) flow cytometer, and lymphocytes were identified by their scatter profile.  The right panel shows confirmation of the specificity by western blot using unconjugated K51-856 antibody at 2.0 μg/ml on lysates from PMA-treated (lane 1, left blot) and control (lane 2) PBMCs and Nocodazole-treated (lane 1, right blot) and control (lane 2) HeLa S3 cells.  Stat1 (pS727) is identified as a band of 91 kDa in the treated cells.

Analysis of Stat1 (pS727) in human epithelioid carcinoma and peripheral blood lymphocytes.  HeLa S3 cells (left panel, ATCC CCL 2.2) were either treated with 0.5 μg/ml Nocodazole (Sigma-Aldrich, Cat. No. M1404) at 37°C for 18 hours (dashed line) or untreated (solid line).  Human peripheral blood mononuclear cells (middle panel) were either stimulated with 50 nM PMA (Sigma, P8139) for 15 minutes at 37ºC (shaded histogram) or untreated (open histogram).  The cells were fixed (BD Cytofix™ buffer, Cat. No. 554655) for 10 minutes at 37°C, permeabilized (BD Phosflow™ Perm Buffer III, Cat. No. 558050) on ice for 30 minutes, and then stained with Alexa Fluor® 647 Mouse anti-Stat1 (pS727, Cat. No. 560190).  Flow cytometry was performed on a BD™ LSR II (left) or FACSCalibur (middle) flow cytometer, and lymphocytes were identified by their scatter profile.  The right panel shows confirmation of the specificity by western blot using unconjugated K51-856 antibody at 2.0 μg/ml on lysates from PMA-treated (lane 1, left blot) and control (lane 2) PBMCs and Nocodazole-treated (lane 1, right blot) and control (lane 2) HeLa S3 cells.  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_1645443
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 to Alexa Fluor® 647 under optimum conditions, and unreacted Alexa Fluor® 647 was removed.

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.

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. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  3. 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.
  4. Alexa Fluor® 647 fluorochrome emission is collected at the same instrument settings as for allophycocyanin (APC).
  5. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  6. The Alexa Fluor®, Pacific Blue™, and Cascade Blue® dye antibody conjugates in this product are sold under license from Molecular Probes, Inc. for research use only, excluding use in combination with microarrays, or as analyte specific reagents. The Alexa Fluor® dyes (except for Alexa Fluor® 430), Pacific Blue™ dye, and Cascade Blue® dye are covered by pending and issued patents.
  7. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
  8. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
560190 Rev. 2
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.

560190 Rev. 2
Format Details
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Alexa Fluor™ 647
Alexa Fluor™ 647 Dye is part of the BD red family of dyes. This is a small organic fluorochrome with an excitation maximum (Ex Max) at 653-nm and an emission maximum (Em Max) at 669-nm. Alexa Fluor 647 is designed to be excited by the Red laser (627-640 nm) and detected using an optical filter centered near 520-nm (e.g., a 660/20 nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
Alexa Fluor™ 647
Red 627-640 nm
653 nm
669 nm
560190 Rev.2
Citations & References
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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
560190 Rev. 2

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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.