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      V450 Mouse anti-Akt (pS473)
      V450 Mouse anti-Akt (pS473)
      Flow cytometric analysis of Akt (pS473) expression in human T-cell leukemia cells.  Human Jurkat cells (ATCC TIB-152) were either treated with 1 µM Wortmannin (Life Technologies, Cat. No. PHZ1301) for 2 hours at 37°C (dotted line histogram) or left untreated (solid line histogram).  The cells were fixed (BD Cytofix™ Fixation 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 BD Phosflow™ V450 Mouse anti-Akt (pS473) (Cat. No. 560858). The data demonstrates that the level of phosphorylation of Akt (pS473) decreases when phosphatidylinositol 3-kinase activity is inhibited by the treatment of Jurkat cells with Wortmannin.  The fluorescence histograms were derived from gated events with the forward and side light-scatter characteristics of intact cells. Flow cytometry was performed on a BD™ LSR II Flow Cytometry System.
      V450 Mouse anti-Akt (pS473)
      Flow cytometric analysis of Akt (pS473) expression in human T-cell leukemia cells.  Human Jurkat cells (ATCC TIB-152) were either treated with 1 µM Wortmannin (Life Technologies, Cat. No. PHZ1301) for 2 hours at 37°C (dotted line histogram) or left untreated (solid line histogram).  The cells were fixed (BD Cytofix™ Fixation 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 BD Phosflow™ V450 Mouse anti-Akt (pS473) (Cat. No. 560858). The data demonstrates that the level of phosphorylation of Akt (pS473) decreases when phosphatidylinositol 3-kinase activity is inhibited by the treatment of Jurkat cells with Wortmannin.  The fluorescence histograms were derived from gated events with the forward and side light-scatter characteristics of intact cells. Flow cytometry was performed on a BD™ LSR II Flow Cytometry System.
      Product Details
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      BD Phosflow™
      Akt1, Akt2, Akt3, PKBα, PKBβ, PKBγ, RAC-PKα, RAC-PKβ, RAC-PKγ, STK-2
      Human (QC Testing), Mouse (Tested in Development)
      Mouse BALB/c IgG1, κ
      Phosphorylated Human Akt1 (pS473) Peptide
      Intracellular staining (flow cytometry) (Routinely Tested)
      5 µl
      AB_10565960
      Aqueous buffered solution containing protein stabilizer 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 BD Horizon™ V450 under optimum conditions, and unreacted BD Horizon™ V450 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. 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.
      5. BD Horizon V450 has a maximum absorption of 406 nm and maximum emission of 450 nm. Before staining with this reagent, please confirm that your flow cytometer is capable of exciting the fluorochrome and discriminating the resulting fluorescence.
      6. Pacific Blue™ is a trademark of Molecular Probes, Inc., Eugene, OR.
      7. All other brands are trademarks of their respective owners.
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      560858 Rev. 2
      Antibody Details
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      M89-61

      Akt [also known as PKB (Protein kinase B) or RAC-PK (Related to the A and C kinases)] is a family of serine/threonine kinases that contains a pleckstrin homology (PH) domain. PH domains play important roles in signal transduction.  There are three known isoforms of Akt in mammalian cells [Akt1 (α), Akt2 (β) and Akt3 (γ)]; they are thought to be regulated similarly.  Akt is activated by insulin and growth factors by a mechanism involving phosphoinositide 3-OH kinase.  Phosphoinositide 3-OH kinases products bind to the PH domain, resulting in translocation of Akt to the plasma membrane and activation of Akt to phospho-Akt by upstream kinases.  Akt is phosphorylated within the activation loop at threonine 308 and the C-terminus at serine 473 (S473).  Phospho-Akt promotes cell survival by inhibiting apoptosis.  Specifically, phospho-Akt1 has been shown to phosphorylate Bad, a member of the Bcl-2 family that promotes cell death.  This phosphorylation results in the inactivation of the proapoptotic function of Bad.  The Akt molecule is thus considered to link extracellular survival signals (growth factors) with the apoptotic machinery (BAD).  Akt is also a key mediator of the metabolic effects of insulin.  Additionally, Akt has been referred to as an oncogene because it has increased activity in a number of tumors.

      The M89-61 antibody recognizes Akt phosphorylated at S473.  This phosphorylation site is shared by all three isoforms of Akt.  The homologous phosphorylation sites in Akt2 and Akt3 are S474 and S472, respectively.

      The antibody is conjugated to BD Horizon™ V450, which has been developed for use in multicolor flow cytometry experiments and is available exclusively from BD Biosciences. It is excited by the Violet laser Ex max of 406 nm and has an Em Max at 450 nm. Conjugates with BD Horizon™ V450 can be used in place of Pacific Blue™ conjugates.

      560858 Rev. 2
      Format Details
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      V450
      BD Horizon™ V450 Dye is part of the BD Horizon™ violet family of dyes. This is a small organic fluorochrome with an excitation maximum (Ex Max) at 405-nm and an emission maximum (Em Max) at 450-nm. BD Horizon™ V450, driven by BD innovation, is designed to be excited by the violet laser (405 nm) and detected using an optical filter centered near 450-nm (e.g., a 450/50-nm bandpass filter). The dye can be excited by the UV (355-nm) laser resulting in cross-laser excitation and spillover. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
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      V450
      Violet 405 nm
      405 nm
      450 nm
      560858 Rev.2
      Citations & References
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      View product citations for antibody "560858" on CiteAb

      Development References (6)

      1. Alessi DR, Andjelkovic M, Caudwell B, et al. Mechanism of activation of protein kinase B by insulin and IGF-1. EMBO J. 1996; 15(23):6541-6551. (Biology). View Reference
      2. Cantley LC, Neel BG. New insights into tumor suppression: PTEN suppresses tumor formation by restraining the phosphoinositide 3-kinase/AKT pathway. Proc Natl Acad Sci U S A. 1999; 96(8):4240-4245. (Biology). View Reference
      3. Datta SR, Dudek H, Tao X, et al. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell. 1997; 91:231-241. (Biology). View Reference
      4. Datta SR, Dudek H, Tao X, et al. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell. 1997; 91:231-241. (Biology). View Reference
      5. Ferrigno P, Silver PA. Regulated nuclear localization of stress-responsive factors: how the nuclear trafficking of protein kinases and transcription factors contributes to cell survival. Oncogene. 1999; 18(45):6129-6134. (Biology). View Reference
      6. Kandel ES, Hay N. The regulation and activities of the multifunctional serine/threonine kinase Akt/PKB. Exp Cell Res. 1999; 253(1):210-229. (Biology). View Reference
      View All (6) View Less
      560858 Rev. 2

      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.