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R718 Mouse Anti-Akt (pS473)
R718 Mouse Anti-Akt (pS473)
Flow cytometric analysis of Akt (pS473) expression in human Jurkat cells. Cells from the human Jurkat (acute T cell leukemia; ATCC TIB-152) cell line were either treated with 1 μM Wortmannin (Life Technologies, Cat. No. PHZ1301) for 2 hours at 37°C (dashed line histogram) or left untreated (solid line histogram). The cells were fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655) for 10 minutes at 37°C, permeabilized with BD Phosflow™ Perm Buffer III (Cat. No. 558050) on ice for 30 minutes, and then stained with BD Phosflow™ R718 Mouse Anti-Akt (pS473) antibody (Cat. No. 567634). The data demonstrate 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 showing Akt (pS473) expression were derived from gated events with the forward and side light-scatter characteristics of intact cells. Flow cytometry and data analysis was performed on a BD™ LSR II Flow Cytometry System and FlowJo™ software.
Flow cytometric analysis of Akt (pS473) expression in human Jurkat cells. Cells from the human Jurkat (acute T cell leukemia; ATCC TIB-152) cell line were either treated with 1 μM Wortmannin (Life Technologies, Cat. No. PHZ1301) for 2 hours at 37°C (dashed line histogram) or left untreated (solid line histogram). The cells were fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655) for 10 minutes at 37°C, permeabilized with BD Phosflow™ Perm Buffer III (Cat. No. 558050) on ice for 30 minutes, and then stained with BD Phosflow™ R718 Mouse Anti-Akt (pS473) antibody (Cat. No. 567634). The data demonstrate 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 showing Akt (pS473) expression were derived from gated events with the forward and side light-scatter characteristics of intact cells. Flow cytometry and data analysis was performed on a BD™ LSR II Flow Cytometry System and FlowJo™ software.
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
Aqueous buffered solution containing ≤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 the dye under optimum conditions and unreacted dye was removed.

Recommended Assay Procedures

BD® CompBeads can be used as surrogates to assess fluorescence spillover (Compensation). When fluorochrome conjugated antibodies are bound to BD® CompBeads, they have spectral properties very similar to cells. However, for some fluorochromes there can be small differences in spectral emissions compared to cells, resulting in spillover values that differ when compared to biological controls. It is strongly recommended that when using a reagent for the first time, users compare the spillover on cells and BD CompBeads to ensure that BD® CompBeads are appropriate for your specific cellular application.

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. An isotype control should be used at the same concentration as the antibody of interest.
  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. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
  5. This product is provided under an Agreement between BIOTIUM and BD Biosciences. This product, and only in the amount purchased by buyer, may be used solely for buyer’s own internal research, in a manner consistent with the accompanying product literature. No other right to use, sell or otherwise transfer (a) this product, or (b) its components is hereby granted expressly, by implication or by estoppel. This product is for research use only. Diagnostic uses require a separate license from Biotium, Inc. For information on purchasing a license to this product including for purposes other than research, contact Biotium, Inc., 3159 Corporate Place, Hayward, CA 94545, Tel: (510) 265-1027. Fax: (510) 265-1352. Email: btinfo@biotium.com.
  6. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
  7. Alexa Fluor™ is a trademark of Life Technologies Corporation.
  8. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
567634 Rev. 1
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 was conjugated to BD Horizon™ Red 718, which has been developed exclusively by for BD Biosciences as a better alternative to Alexa Fluor™ 700. BD Horizon™ Red 718 can be excited by the red laser (628 – 640 nm) and, with an Em Max around 718 nm, it can be detected using a 730/45 nm filter. Due to similar excitation and emission properties, we do not recommend using R718 in combination with APC-R700 or Alexa Fluor™ 700.

567634 Rev. 1
Format Details
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R718
The BD Horizon™ Red 718 (R718) Dye is part of the BD red family of dyes. It is a small organic fluorochrome with an excitation maximum (Ex Max) at 695-nm and an emission maximum (Em Max) at 718-nm. Driven by BD innovation, R718 is designed to be excited by the red laser (627–640-nm) and detected using an optical filter centered near 720-nm (e.g., a 720/40-nm bandpass filter). R718 is a brighter alternative to Alexa Fluor™ 700. R718 is also a bright small molecule alternative to APC-R700 with lower spread into the APC detector. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
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R718
Red 627-640 nm
695 nm
718 nm
567634 Rev.1
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
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567634 Rev. 1

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For Research Use Only. Not for use in diagnostic or therapeutic procedures.