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      Alexa Fluor® 647 Mouse anti-Btk (pY551)/Itk (pY511)
      Alexa Fluor® 647 Mouse anti-Btk (pY551)/Itk (pY511)
      Analysis of Btk (pY551) in activated human B lymphoma cells.  Ramos cells (ATCC CRL-1596) were serum starved overnight and then either stimulated with 5 mM hydrogen peroxide for 15 minutes (shaded histogram) or unstimulated (open histogram).  The cells were fixed (BD Cytofix™ Fixation 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-Btk (pY551)/Itk (pY511). Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.
      Alexa Fluor® 647 Mouse anti-Btk (pY551)/Itk (pY511)
      Analysis of Btk (pY551) in activated human B lymphoma cells.  Ramos cells (ATCC CRL-1596) were serum starved overnight and then either stimulated with 5 mM hydrogen peroxide for 15 minutes (shaded histogram) or unstimulated (open histogram).  The cells were fixed (BD Cytofix™ Fixation 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-Btk (pY551)/Itk (pY511). Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.
      Product Details
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      BD Phosflow™
      Human (QC Testing)
      Mouse IgG1, κ
      Phosphorylated Human Btk Peptide
      Intracellular staining (flow cytometry) (Routinely Tested)
      20 µl
      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. 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.
      4. Alexa Fluor® 647 fluorochrome emission is collected at the same instrument settings as for allophycocyanin (APC).
      5. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
      6. 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.
      7. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
      8. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
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      558134 Rev. 3
      Antibody Details
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      24a/BTK (Y551)

      Bruton's tyrosine kinase (Btk) is a nonreceptor tyrosine kinase whose function is critical for proper B cell development and signaling.  The activity of Btk is regulated by Src mediated phosphorylation of the kinase domain at tyrosine 551 (Y551).  This event induces Btk kinase activity and subsequent autophosphorylation at Y223.  Phosphorylated Btk then associates with the cell membrane via the interaction of the PH domain with phosphatidylinositol 3, 4, 5-triphosphate.  

      The Tec family kinase Itk plays a critical role in signal transduction downstream of the T cell antigen receptor and has been implicated in the activation of phospholipase C-γ1 (PLCγ1), a key regulator of calcium mobilization and extracellular signal-regulated kinase (ERK) activation.  Itk is regulated by an activating transphosphorylation event in which Y511 in the kinase domain is phosphorylated by Lck.  

      The 24a/BTK (Y551) monoclonal antibody recognizes the Y551-phosphorylated form of human Btk and the Y511 phosphorylated form of human Itk.  

      558134 Rev. 3
      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
      558134 Rev.3
      Citations & References
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      View product citations for antibody "558134" on CiteAb

      Development References (3)

      1. Mahajan S, Fargnoli J, Burkhardt AL, Kut SA, Saouaf SJ, Bolen JB. Src family protein tyrosine kinases induce autoactivation of Bruton's tyrosine kinase. Mol Cell Biol. 1995; 15:5304-5311. (Biology). View Reference
      2. Rawlings DJ, Scharenberg AM, Park H, et al. Activation of BTK by a phosphorylation mechanism initiated by SRC family kinases. Science. 1996; 271:822-825. (Biology). View Reference
      3. Wilcox HM, Berg LJ.. Itk phosphorylation sites are required for functional activity in primary T cells. J Biol Chem. 2003; 278:37112-37121. (Biology).
      558134 Rev. 3

       

      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.