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Alexa Fluor® 488 Mouse anti-4EBP1 (pT69)
Alexa Fluor® 488 Mouse anti-4EBP1 (pT69)

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, left panel).  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® 488 Mouse anti-4EBP1 (pT69, Cat. No. 560290).  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.

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. 612656/612657) was the gel-loading control.

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, left panel).  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® 488 Mouse anti-4EBP1 (pT69, Cat. No. 560290).  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.

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. 612656/612657) was the gel-loading control.

Product Details
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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_1645340
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® 488 under optimum conditions, and unreacted Alexa Fluor® 488 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

  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® 488 fluorochrome emission is collected at the same instrument settings as for fluorescein isothiocyanate (FITC).
  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.
560290 Rev. 2
Antibody Details
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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.  

560290 Rev. 2
Format Details
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Alexa Fluor™ 488
Alexa Fluor™ 488 Dye is part of the BD blue family of dyes. This is a small organic fluorochrome with an excitation maximum (Ex Max) at 494-nm and an emission maximum (Em Max) at 517-nm. Alexa Fluor™ 488 is designed to be excited by the Blue laser (488 nm) and detected using an optical filter centered near 520-nm (e.g., a 530/30-nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
Alexa Fluor™ 488
Blue 488 nm
494 nm
517 nm
560290 Rev.2
Citations & References
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Development References (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). View Reference
  2. Hay N, Sonenberg N. Upstream and downstream of mTOR. Genes Dev. 2004; 18:1926-1945. (Biology).
560290 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.