BD Phosflow™ R718 Mouse Anti-Akt (pS473)
Clone M89-61 (RUO)
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Preparation And Storage
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
- 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).
- An isotype control should be used at the same concentration as the antibody of interest.
- 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.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
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- Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
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.
Development References (8)
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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
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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
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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
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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
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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
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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
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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
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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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For Research Use Only. Not for use in diagnostic or therapeutic procedures.