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PE Mouse anti-Akt1
PE Mouse anti-Akt1

Analysis of Akt1 in human epithelioid carcinoma.  HeLa S3 cells (ATCC CCL 2.2) were either transfected with Akt1 RNAi (open histogram) or untreated (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 30 minutes, and then stained with PE Mouse anti-Akt1.  Down-regulation of Akt1 expression is evident in the RNAi-transfected cells.  Flow cytometry was performed on a BD FACSArray™ bioanalyzer system.

Analysis of Akt1 in human epithelioid carcinoma.  HeLa S3 cells (ATCC CCL 2.2) were either transfected with Akt1 RNAi (open histogram) or untreated (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 30 minutes, and then stained with PE Mouse anti-Akt1.  Down-regulation of Akt1 expression is evident in the RNAi-transfected cells.  Flow cytometry was performed on a BD FACSArray™ bioanalyzer system.

Product Details
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BD Phosflow™
PKBα, RAC-PK
Human (QC Testing), Human, Mouse, Rat, Dog (Reactivity Confirmed in Development)
Mouse IgG1, κ
Human Akt1 Recombinant Protein
Intracellular staining (flow cytometry) (Routinely Tested)
20 µl
AB_1645500
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 with R-PE under optimum conditions, and unconjugated antibody and free PE were removed.

Recommended Assay Procedures

Either BD Cytofix™ fixation buffer or BD Phosflow™ Fix Buffer I may be used for cell fixation.

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. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  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. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  5. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
560049 Rev. 2
Antibody Details
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55/PKBa/Akt

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 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 55/PKBa/Akt monoclonal antibody recognizes Akt1, regardless of phosphorylation status.

The specificity of this antibody conjugate for flow cytometric analysis was validated by confirming that RNA-mediated interference (RNAi) of the specific protein reduced the staining of the cells (see figure).  Furthermore, the capacity of the RNAi to down-regulate the expression of the relevant protein was confirmed by western blot analysis.

560049 Rev. 2
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
560049 Rev.2
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: Activation). 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. 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
560049 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.

Refer to manufacturer's instructions for use and related User Manuals and Technical Data Sheets before using this product 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.