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Alexa Fluor® 647 Mouse anti-4EBP1 (pT69)
Alexa Fluor® 647 Mouse anti-4EBP1 (pT69)

LEFT PANEL: Analysis of 4EBP1 (pT69) in human peripheral blood monocytes. Human peripheral blood mononuclear cells (PBMC) were either treated with 100 μM LY294002 (Sigma, Cat. No. L-9908) for 1 hour at 37ºC (shaded histogram) or untreated (open histogram).  The PBMC were fixed (BD Cytofix™ buffer, Cat. No. 554655) for 10 minutes at 37°C, permeabilized with BD Phosflow™ Perm Buffer III (Cat. No. 558050) on ice for 30 minutes, and then stained with Alexa Fluor® 647 Mouse anti-4EBP1 (pT69)(Cat. No. 560289).  For data analysis, monocytes were selected by their scatter profile.  The data demonstrates that the level of phosphorylation of 4EBP1 decreases when protein kinase activity is inhibited by the treatment.  Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.

RIGHT PANEL: The specificity of mAb M34-273 was confirmed by western blot analysis (right panel) using unconjugated polyclonal anti-4EBP1 (Cell Signaling Technology, Cat. No. 9542, left blot) and unconjugated monoclonal Mouse anti-4EBP1 (pT69) (right blot) antibodies on lysates from control (lanes 1) and LY294002-treated (lanes 2) PBMC.  4EBP1 is identified as a band of 15-20 kDa in the left blot, regardless of LY294002 treatment.  The right blot demonstrates the reduction of 4EBP1 (pT69) with LY294002 treatment (lane 2).  Purified Mouse anti-Actin monoclonal antibody (Cat. No. 612656 or 612657) was the gel-loading control.

LEFT PANEL: Analysis of 4EBP1 (pT69) in human peripheral blood monocytes. Human peripheral blood mononuclear cells (PBMC) were either treated with 100 μM LY294002 (Sigma, Cat. No. L-9908) for 1 hour at 37ºC (shaded histogram) or untreated (open histogram).  The PBMC were fixed (BD Cytofix™ buffer, Cat. No. 554655) for 10 minutes at 37°C, permeabilized with BD Phosflow™ Perm Buffer III (Cat. No. 558050) on ice for 30 minutes, and then stained with Alexa Fluor® 647 Mouse anti-4EBP1 (pT69)(Cat. No. 560289).  For data analysis, monocytes were selected by their scatter profile.  The data demonstrates that the level of phosphorylation of 4EBP1 decreases when protein kinase activity is inhibited by the treatment.  Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.

RIGHT PANEL: The specificity of mAb M34-273 was confirmed by western blot analysis (right panel) using unconjugated polyclonal anti-4EBP1 (Cell Signaling Technology, Cat. No. 9542, left blot) and unconjugated monoclonal Mouse anti-4EBP1 (pT69) (right blot) antibodies on lysates from control (lanes 1) and LY294002-treated (lanes 2) PBMC.  4EBP1 is identified as a band of 15-20 kDa in the left blot, regardless of LY294002 treatment.  The right blot demonstrates the reduction of 4EBP1 (pT69) with LY294002 treatment (lane 2).  Purified Mouse anti-Actin monoclonal antibody (Cat. No. 612656 or 612657) was the gel-loading control.

商品详情
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BD Phosflow™
4E-BP1. EIF4EBP1, P/OKCL.6, PHAS-I, PHAS-1
Human (QC Testing)
Mouse BALB/c IgG1, κ
Phosphorylated Human 4EBP1 (pT69) Peptide
Intracellular staining (flow cytometry) (Routinely Tested)
20 µl
AB_1645395
Aqueous buffered solution containing BSA and ≤0.09% sodium azide.
RUO


准备和存储

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. Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.

推荐的实验流程

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.

商品通知

  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. Alexa Fluor® 647 fluorochrome emission is collected at the same instrument settings as for allophycocyanin (APC).
  5. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  6. 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.
  7. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
  8. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
560289 Rev. 2
抗体详情
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M34-273

The eukaryotic translation initiation factor 4E-Binding Protein 1 (4EBP1) is a phosphorylated heat- and acid-stable protein (PHAS-I or PHAS-1), and it is regulated by insulin. It is a member of the eIF4E-Binding Protein Family, which also includes the proteins 4EBP2 and 4EBP3. 4EBP1 binds with eukaryotic translation Initiation Factor 4E (eIF4E), which prevents its assembly into the eIF4E complex and inhibits cap-dependent translation. When 4EBP1 is phosphorylated, this binding is disrupted, allowing cap-dependent translation to be activated. Phosphorylation of 4EBP1 is required for protein synthesis, and it mediates the regulation of protein translation by stimuli that signal through the phosphoinositide 3 (PI3) kinase pathway. We found that threonine 69 (T69) is phosphorylated in resting human peripheral blood monocytes, but it is almost undetectable in resting lymphocytes. PI3 kinase inhibitors, such as LY294002 down-regulate the phosphorylation level of 4EBP1 (pT69) in monocytes.

The M34-273 monoclonal antibody recognizes the phosphorylated T69 of activated human 4EBP1.  

560289 Rev. 2
格式详情
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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 520-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
Red 627-640 nm
653 nm
669 nm
560289 Rev.2
报价单和参考
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研发参考 (2)

  1. Gingras AC, Raught B, Gygi SP, et al. Hierarchical phosphorylation of the translation inhibitor 4E-BP1. Genes Dev. 2001; 15(21):2852-2864. (Biology). 查看参考
  2. Hay N, Sonenberg N. Upstream and downstream of mTOR. Genes Dev. 2004; 18:1926-1945. (Biology).
560289 Rev. 2

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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.