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PE Mouse Anti-Akt (pT308)
PE Mouse Anti-Akt (pT308)
Analysis of Akt (pT308) in activated mouse embryonic fibroblasts.  NIH/3T3 cells (ATCC CR:-1658) were serum starved overnight and either stimulated with Platelet-Derived Growth Factor-BB (Cat. No. 354051) at 37˚C for 30 minutes (filled histogram) or unstimulated (open histogram).  The cells were fixed with BD Phosflow™ Fix Buffer I (Cat. No. 557870) at 37˚C for 10 minutes, permeabilized in BD Phosflow™ Perm Buffer III (Cat. No. 558050) on ice for at least 30 minutes (or overnight at -20˚C), and stained with PE anti-Akt (pT308, Cat. No. 558275).  Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.
Analysis of Akt (pT308) in activated mouse embryonic fibroblasts.  NIH/3T3 cells (ATCC CR:-1658) were serum starved overnight and either stimulated with Platelet-Derived Growth Factor-BB (Cat. No. 354051) at 37˚C for 30 minutes (filled histogram) or unstimulated (open histogram).  The cells were fixed with BD Phosflow™ Fix Buffer I (Cat. No. 557870) at 37˚C for 10 minutes, permeabilized in BD Phosflow™ Perm Buffer III (Cat. No. 558050) on ice for at least 30 minutes (or overnight at -20˚C), and stained with PE anti-Akt (pT308, Cat. No. 558275).  Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.
Product Details
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BD Phosflow™
AKT; PKB; PKB alpha; PKB-ALPHA; PRKBA; RAC; RAC-ALPHA; RAC-PK-alpha
Mouse (QC Testing), Human (Tested in Development)
Mouse IgG1, κ
Phosphorylated Human Akt1 Peptide
Intracellular staining (flow cytometry) (Routinely Tested)
20 µl
207, 11651
AB_2225329
Aqueous buffered solution containing BSA and ≤0.09% sodium azide.
RUO


Preparation And Storage

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 unconjugated antibody and free dye were removed. Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.

Recommended Assay Procedures

This antibody detects Akt (pT308) in human and mouse cell lines.  We have been unable to detect upregulated phosphorylation of Akt in activated leukocytes, such as whole blood and peripheral blood mononuclear cells.

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 cell and BD® CompBeads to ensure that BD® CompBeads are appropriate for your specific cellular application.

Product Notices

  1. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
  2. 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).
  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. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  5. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  6. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
  7. For U.S. patents that may apply, see bd.com/patents.
558275 Rev. 6
Antibody Details
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J1-223.371

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 kinase 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 (T308) and the C-terminus at serine 473.  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 J1-223.371 antibody recognizes Akt phosphorylated at T308.

558275 Rev. 6
Format Details
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PE
R-Phycoerythrin (PE), is part of the BD family of Phycobiliprotein dyes. This fluorochrome is a multimeric fluorescent phycobiliprotein with excitation maximum (Ex Max) of 496 nm and 566 nm and an emission maximum (Em Max) at 576 nm. PE is designed to be excited by the Blue (488 nm), Green (532 nm) and Yellow-Green (561 nm) lasers and detected using an optical filter centered near 575 nm (e.g., a 575/26-nm bandpass filter). As PE is excited by multiple lasers, this can result in cross-laser excitation and fluorescence spillover on instruments with various combinations of Blue, Green, and Yellow-Green lasers. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
altImg
PE
Yellow-Green 488 nm, 532 nm, 561 nm
496 nm, 566 nm
576 nm
558275 Rev.6
Citations & References
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Development References (5)

  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. De Falco E, et al.. Altered SDF-1-mediated differentiation of bone marrow-derived endothelial progenitor cells in diabetes mellitus.. J Cell Mol Med. 2009; 13(9B):3405-3414. (Clone-specific: Flow cytometry). 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. 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 (5) View Less
558275 Rev. 6

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.