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      Alexa Fluor® 647 Mouse Anti-Stat3 (pS727)
      Alexa Fluor® 647 Mouse Anti-Stat3 (pS727)
      Analysis of Stat3 (pS727) in monocytes.  Human peripheral blood mononuclear cells (PBMC) were either stimulated with 40 nM PMA (Sigma, P8139) for 15 minutes (shaded histogram) or unstimulated (open histogram).  The cells were fixed (BD Cytofix™ buffer, Cat. No. 554655) for 10 minutes, then permeabilized (BD Phosflow™ Perm Buffer III, Cat. No. 558050) on ice for at least 30 minutes, and then stained with Alexa Fluor® 647 anti-Stat3 (pS727) (Cat. No. 558099).  Monocytes were selected by scatter profile.  Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.
      Alexa Fluor® 647 Mouse Anti-Stat3 (pS727)
      Analysis of Stat3 (pS727) in monocytes.  Human peripheral blood mononuclear cells (PBMC) were either stimulated with 40 nM PMA (Sigma, P8139) for 15 minutes (shaded histogram) or unstimulated (open histogram).  The cells were fixed (BD Cytofix™ buffer, Cat. No. 554655) for 10 minutes, then permeabilized (BD Phosflow™ Perm Buffer III, Cat. No. 558050) on ice for at least 30 minutes, and then stained with Alexa Fluor® 647 anti-Stat3 (pS727) (Cat. No. 558099).  Monocytes were selected by scatter profile.  Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.
      Product Details
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      BD Phosflow™
      Human (QC Testing), Mouse,Rat (Tested in Development)
      Mouse IgG1
      Phosphorylated Human Stat3
      Intracellular staining (flow cytometry) (Routinely Tested)
      20 µl
      AB_397024
      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.
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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. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
      3. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
      4. 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.
      5. Alexa Fluor® 647 fluorochrome emission is collected at the same instrument settings as for allophycocyanin (APC).
      6. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
      7. 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.
      8. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
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      558099 Rev. 5
      Antibody Details
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      49/p-Stat3

      The 49/p-Stat3 monoclonal antibody recognizes the S727-phosphorylated form of Signal transducer and activator of transcription 3 (Stat3/STAT3 isoform 1).  The fluorochrome-conjugated formats have been evaluated using a human model system. However, the unconjugated form of this antibody is also effective for western blot analysis of human, mouse, and rat tissue. The Stat proteins function both as cytoplasmic signal transducers and as activators of transcription.  Seven mammalian Stat proteins have been identified: Stat1-4, Stat5a, 5b, and Stat6. Stat3 is a 92-kDa protein that is activated as a DNA binding protein through cytokines, such as IL-6, and growth factors, such as EGF.  Stat3 is phophorylated at serine 727 (S727) via the MAPK pathway.  The S727 residue is located at a conserved Pro-X-Ser-Pro sequence, which is recognized by the protein kinase ERK.  Activation through the S727 residue is thought to lead to initiation of transcription.  Upon activation, Stat3 dimerizes, translocates to the nucleus, and binds DNA response elements thereby regulating gene expression.  It appears that Stat3 binds to DNA as a homodimer, but it is also capable of binding as a heterodimer with Stat1.  In addition to serine phosphorylation, Stat3 is also phosphorylated at tyrosine 705 by JAK1 in response to cytokine stimulation.  Stat3 is widely expressed and can bind to the sis-inducible element (SIE) site from the c-fos promoter. This site is similar to the GAS element that is present in IFN--induced genes. Thus, phosphorylation of S727 in Stat3 occurs in response to growth factors and cytokines, and is essential for normal transcriptional activity.

      558099 Rev. 5
      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 670-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
      558099 Rev.5
      Citations & References
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      Development References (4)

      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. Imada K, Leonard WJ. The Jak-STAT pathway. Mol Immunol. 2000; 37:1-11. (Biology). View Reference
      3. Kanai M, Konda Y, Nakajima T, et al . Differentiation-inducing factor-1 (DIF-1) inhibits STAT3 activity involved in gastric cancer cell proliferation via MEK-ERK-dependent pathway. Oncogene. 2003; 22(22):548-554. (Biology). View Reference
      4. Schuringa JJ, Dekker LV, Vellenga E, Kruijer W. Sequential activation of Rac-1, SEK-1/MKK-4, and protein kinase Cδ is required for interleukin-6-induced STAT3 Ser-727 phosphorylation and transactivation. J Biol Chem. 2001; 276:27709-27715. (Biology).
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      558099 Rev. 5

      Please refer to Support Documents for Quality Certificates


      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.

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