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PE Mouse anti-elF4E (pS209)
PE Mouse anti-elF4E (pS209)
Analysis of eIF4E (pS209) in monocytes.  Human peripheral blood mononuclear cells (PBMC) were either stimulated with 40 nM Phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich Cat. No. P8139) for 15 minutes (shaded histogram) or unstimulated (open histogram).  The cells were fixed (BD Cytofix™ buffer, Cat. No. 554655) for 10 minutes, then permeabilized (BD Phosflow™ Perm Buffer II, Cat. No. 558052) on ice for at least 30 minutes, and then stained with PE Mouse anti-elF4E (pS209, Cat. No. 560229).  Monocytes were selected by scatter profile.  Flow cytometry was performed on a BD FACSArray™ bioanalyzer system. The specificity of mAb J77-925 was confirmed by western blot analysis using unconjugated antibody on lysates from control (lane 1) and PMA-treated (lane 2) PBMC.  elF4E (pS209) is identified as a band of 25 kDa, with increased intensity in the treated lane.
Analysis of eIF4E (pS209) in monocytes.  Human peripheral blood mononuclear cells (PBMC) were either stimulated with 40 nM Phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich Cat. No. P8139) for 15 minutes (shaded histogram) or unstimulated (open histogram).  The cells were fixed (BD Cytofix™ buffer, Cat. No. 554655) for 10 minutes, then permeabilized (BD Phosflow™ Perm Buffer II, Cat. No. 558052) on ice for at least 30 minutes, and then stained with PE Mouse anti-elF4E (pS209, Cat. No. 560229).  Monocytes were selected by scatter profile.  Flow cytometry was performed on a BD FACSArray™ bioanalyzer system. The specificity of mAb J77-925 was confirmed by western blot analysis using unconjugated antibody on lysates from control (lane 1) and PMA-treated (lane 2) PBMC.  elF4E (pS209) is identified as a band of 25 kDa, with increased intensity in the treated lane.
Product Details
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BD Phosflow™
eIF-4E, CBP, EIF4E1, EIF4EL1, EIF4F, mRNA cap-binding protein
Human (QC Testing), Mouse, Rat (Predicted)
Mouse IgG1, κ
Phosphorylated Human eIF4E Peptide
Intracellular staining (flow cytometry) (Routinely Tested)
20 µl
AB_1645504
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.  Any of the three BD Phosflow™ permeabilization buffers may be used.

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. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  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. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
560229 Rev. 2
Antibody Details
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J77-925

The eukaryotic translation Initiation Factor 4E (eIF4E) is a 25-kDa phosphoprotein that specifically binds to the 7-methylguanosine-containing cap of mRNA.  eIF4E is the rate-limiting component for the initiation of cap-dependent translation by the eIF4F translation initiation complex, which is composed of eIF4E, eIF4G, and eIF4A.  This complex promotes the unwinding of secondary structure at the 5' untranslated region of mRNA that is necessary to expose and locate the AUG initiation codon.  Other functions of eIF4E have been identified, such as promoting the export of mRNAs that are involved in cell cycle progression from the nucleus and differentially regulating the translation of certain mRNAs in the cytoplasm.  Three mechanisms for eIF4E regulation have been identified:  Mnk1-mediated phosphorylation on serine 209 (S209) is required for eIF4E binding to the cap structure; over-expression of phosphorylated eIF4E can lead to increased cell proliferation, suppression of apoptosis, and a transformed phenotype; and interactions with nonphosphorylated eIF4E-binding proteins inhibit the formation of the eIF4F complex.  

The J77-925 monoclonal antibody recognizes the phosphorylated S209 (pS209) of eIF4E.

560229 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.
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PE
Yellow-Green 488 nm, 532 nm, 561 nm
496 nm, 566 nm
576 nm
560229 Rev.2
Citations & References
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Development References (5)

  1. Culjkovic B, Topisirovic I, Borden KLB. Controlling gene expression through RNA regulons. The role of the eukaryotic translation initiation factor eIF4E. Cell Cycle. 2007; 6(1):65-69. (Biology).
  2. Grolleau A, Kaplan MJ, Hanash SM, Beretta L, Richardson B. Impaired translational response and increased protein kinase PKR expression in T cells from lupus patients. J Clin Invest. 2000; 106(12):1561-1568. (Biology).
  3. Mendez R, Myers Jr MG, White MF, Rhoads RE. Stimulation of protein synthesis, eukaryotic translation initiation factor 4E phosphorylation, and PHAS-I phosphorylation by insulin requires insulin receptor substrate 1 and phosphatidylinositol 3-kinase. Mol Cell Biol. 1996; 16(6):2857-2864. (Biology). View Reference
  4. Waskiewicz AJ, Flynn A, Proud CG, Cooper JA. Mitogen-activated protein kinases activate the serine/threonine kinases Mnk1 and Mnk2. EMBO J. 1997; 16(8):1909-1920. (Biology). View Reference
  5. Wendel H-G, Silva RLA, Malina A, et al. Dissecting eIF4E action in tumorigenesis. Genes Dev. 2007; 21:3232-3237. (Biology).
View All (5) View Less
560229 Rev. 2

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