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BD Phosflow™ Alexa Fluor® 647 Mouse anti-elF4E (pS209)
Clone J77-925 (RUO)
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 Alexa Fluor® 647 Mouse anti-elF4E (pS209, Cat. No. 560240). Monocytes were selected by scatter profile. Flow cytometry was performed on a BD FACSArray™ bioanalyzer system.
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 Alexa Fluor® 647 Mouse anti-elF4E (pS209, Cat. No. 560240). Monocytes were selected by scatter profile. Flow cytometry was performed on a BD FACSArray™ bioanalyzer system.
The specificity of Alexa Fluor® 647 Mouse anti-elF4E(pS209) was confirmed by western blot analysis. Lysates from control populations (left blot) and PMA-treated populations (right blot) were treated with unconjugated antibody. ElF4E (pS209) is identified as a band of 25 kDa, with increased intensity in the treated cells.
The specificity of Alexa Fluor® 647 Mouse anti-elF4E(pS209) was confirmed by western blot analysis. Lysates from control populations (left blot) and PMA-treated populations (right blot) were treated with unconjugated antibody. ElF4E (pS209) is identified as a band of 25 kDa, with increased intensity in the treated cells.
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 Alexa Fluor® 647 Mouse anti-elF4E (pS209, Cat. No. 560240). Monocytes were selected by scatter profile. Flow cytometry was performed on a BD FACSArray™ bioanalyzer system.
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 Alexa Fluor® 647 Mouse anti-elF4E (pS209, Cat. No. 560240). Monocytes were selected by scatter profile. Flow cytometry was performed on a BD FACSArray™ bioanalyzer system.
The specificity of Alexa Fluor® 647 Mouse anti-elF4E(pS209) was confirmed by western blot analysis. Lysates from control populations (left blot) and PMA-treated populations (right blot) were treated with unconjugated antibody. ElF4E (pS209) is identified as a band of 25 kDa, with increased intensity in the treated cells.
The specificity of Alexa Fluor® 647 Mouse anti-elF4E(pS209) was confirmed by western blot analysis. Lysates from control populations (left blot) and PMA-treated populations (right blot) were treated with unconjugated antibody. ElF4E (pS209) is identified as a band of 25 kDa, with increased intensity in the treated cells.
Product Details
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_1645408
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 to Alexa Fluor® 647 under optimum conditions, and unreacted Alexa Fluor® 647 was 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
- 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).
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- 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.
- Alexa Fluor® 647 fluorochrome emission is collected at the same instrument settings as for allophycocyanin (APC).
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- The Alexa Fluor®, Pacific Blue™, and Cascade Blue® dye antibody conjugates in this product are sold under license from Molecular Probes, Inc. for research use only, excluding use in combination with microarrays, or as analyte specific reagents. The Alexa Fluor® dyes (except for Alexa Fluor® 430), Pacific Blue™ dye, and Cascade Blue® dye are covered by pending and issued patents.
- Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
Companion Products
Stain Buffer (FBS) RUO
Size
500 ML
Cat No.
554656
Stain Buffer (BSA) RUO
Size
500 ML
Cat No.
554657
Fixation Buffer RUO
Size
100 ML
Cat No.
554655
Fix Buffer I RUO
Size
250 ML
Cat No.
557870
Perm/Wash Buffer I RUO
Size
125 ML
Cat No.
557885
Perm Buffer II RUO
Size
125 ML
Cat No.
558052
560240 Rev. 2
Antibody Details
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.
560240 Rev. 2
Format Details
Alexa Fluor™ 647
Alexa Fluor™ 647 Dye is part of the BD red family of dyes. This is a small organic fluorochrome with an excitation maximum (Ex Max) at 653-nm and an emission maximum (Em Max) at 669-nm. Alexa Fluor™ 647 is designed to be excited by the Red laser (627-640 nm) and detected using an optical filter centered near 670-nm (e.g., a 660/20 nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
Alexa Fluor™ 647
653 nm
669 nm
560240 Rev.2
Citations & References
View product citations for antibody "560240" on CiteAb
Development References (5)
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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).
-
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).
-
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
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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
-
Wendel H-G, Silva RLA, Malina A, et al. Dissecting eIF4E action in tumorigenesis. Genes Dev. 2007; 21:3232-3237. (Biology).
560240 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.
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.