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Alexa Fluor® 647 Mouse anti-Akt (pS473)
Alexa Fluor® 647 Mouse anti-Akt (pS473)

LEFT: Analysis of Akt (pS473) in mouse embryonic fibroblasts.  Serum-starved NIH/3T3 cells (ATCC CRL-1658) were either stimulated with PDGF-BB (Cat. No. 354051, open histogram) or unstimulated (shaded 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 Alexa Fluor® 647 Mouse anti-Akt (pS473). Flow cytometry was performed on a BD FACSArray™ flow cytometry system.

RIGHT: Analysis of Akt (pS473) in human T-cell leukemia.  Jurkat cells (ATCC TIB-152) were either treated with 1 µM Wortmannin (Invitrogen, Cat. No. PHZ1301) for 2 hours at 37°C (shaded histogram) or left untreated (open 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 Alexa Fluor® 647 Mouse anti-Akt (pS473). The data demonstrates that the level of phosphorylation of Akt (pS473) decreases when phosphatidylinositol 3-kinase activity is inhibited by the treatment.  Flow cytometry was performed on a BD FACSArray™ flow cytometry system.

LEFT: Analysis of Akt (pS473) in mouse embryonic fibroblasts.  Serum-starved NIH/3T3 cells (ATCC CRL-1658) were either stimulated with PDGF-BB (Cat. No. 354051, open histogram) or unstimulated (shaded 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 Alexa Fluor® 647 Mouse anti-Akt (pS473). Flow cytometry was performed on a BD FACSArray™ flow cytometry system.

RIGHT: Analysis of Akt (pS473) in human T-cell leukemia.  Jurkat cells (ATCC TIB-152) were either treated with 1 µM Wortmannin (Invitrogen, Cat. No. PHZ1301) for 2 hours at 37°C (shaded histogram) or left untreated (open 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 Alexa Fluor® 647 Mouse anti-Akt (pS473). The data demonstrates that the level of phosphorylation of Akt (pS473) decreases when phosphatidylinositol 3-kinase activity is inhibited by the treatment.  Flow cytometry was performed on a BD FACSArray™ 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)
20 µl
AB_1645397
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.

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. Alexa Fluor® 647 fluorochrome emission is collected at the same instrument settings as for allophycocyanin (APC).
  4. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  5. 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.
  6. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
  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. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
560343 Rev. 3
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.

560343 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
560343 Rev.3
Citations & References
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Development References (8)

  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. 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
  5. Herishanu Y, Kay S, Dezorella N, et al. Divergence in CD19-mediated signaling unfolds intraclonal diversity in chronic lymphocytic leukemia, which correlates with disease progression. J Immunol. 2013; 190(2):784-793. (Clone-specific: Flow cytometry). 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
  7. Prinz PU, Mendler AN, Masouris I, Durner L, Oberneder R, Noessner E. High DGK-alpha and disabled MAPK pathways cause dysfunction of human tumor-infiltrating CD8+ T cells that is reversible by pharmacologic intervention. J Immunol. 2012; 188(12):5990-6000. (Clone-specific: Flow cytometry). View Reference
  8. Schlickeiser S, Stanojlovic S, Appelt C, et al. Control of TNF-induced dendritic cell maturation by hybrid-type N-glycans. J Immunol. 2011; 186(9):5201-5211. (Clone-specific: Flow cytometry). View Reference
View All (8) View Less
560343 Rev. 3

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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.