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FITC Mouse Anti- eIF-4E
FITC Mouse Anti- eIF-4E

Immunofluorescent staining of HeLa cells.  HeLa cells (Human cervical epitheloid carcinoma; ATCC CCL-2.2) were seeded in a 96-well imaging plate at ~ 10,000 cells per well.  After overnight incubation, cells were stained using the methanol fix/perm protocol (see Recommended Assay Procedure) and the FITC anti- eIF-4E antibody (Cat. No. 611624).  Images were taken on a Pathway 855 imager using a 20x objective.  This antibody also stained U2OS cells.  The Triton-X 100 fix/perm protocol (see Recommended Assay Procedure) is not recommended for use with this antibody.

Immunofluorescent staining of HeLa cells.  HeLa cells (Human cervical epitheloid carcinoma; ATCC CCL-2.2) were seeded in a 96-well imaging plate at ~ 10,000 cells per well.  After overnight incubation, cells were stained using the methanol fix/perm protocol (see Recommended Assay Procedure) and the FITC anti- eIF-4E antibody (Cat. No. 611624).  Images were taken on a Pathway 855 imager using a 20x objective.  This antibody also stained U2OS cells.  The Triton-X 100 fix/perm protocol (see Recommended Assay Procedure) is not recommended for use with this antibody.

Product Details
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BD Transduction Laboratories™
Human (QC Testing), Mouse, Rat, Dog, Chicken, Frog (Tested in Development)
Mouse IgG1
Rabbit eIF-4E aa. 1-217
Immunofluorescence (Routinely Tested), Bioimaging (Tested During Development)
25 kda
250 µg/ml
AB_399066
Aqueous buffered solution containing BSA, glycerol, and ≤0.09% sodium azide.
RUO


Preparation And Storage

The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated with FITC under optimum conditions, and unreacted FITC was removed. Store undiluted at -20°C.

Recommended Assay Procedures

Bioimaging:  For reccommended Bioimaging protocol, please refer to the protocols section of "Cellular Imaging" at our website, http://www.bdbiosciences.com/us/s/resources. Note that this antibody has been developed and certified for the bioimaging application. However, a routine bioimaging test is not performed on every lot. Researchers are encouraged to titrate the reagent for optimal performance.

Methanol Procedure for a 96 well plate:

Remove media from wells. Add 100 µl/well fresh 3.7% Formaldehyde in PBS. Incubate for 10 minutes at room temperature (RT). Flick out and add 100 µl/well 90% methanol. Incubate for 5 minutes at RT. Flick out and wash twice with PBS. Flick out PBS and add 100 µl/well blocking buffer (3% FBS in PBS). Incubate for 30 minutes at RT. Flick out and add diluted antibody (diluted in blocking buffer). Incubate for 1 hour at RT. Wash three times with PBS. Flick out PBS and add second step reagent. Incubate for 1 hour at RT. Wash three times with PBS. Image sample.

Triton-X 100 Procedure for a 96 well plate:

Remove media from wells. Add 100 µl/well fresh 3.7% Formaldehyde in PBS. Incubate for 10 minutes at room temperature (RT). Flick out and add 100 µl/well 0.1% Triton-X 100. Incubate for 5 minutes at RT. Flick out and wash twice with PBS. Flick out PBS and add 100 µl/well blocking buffer (3% FBS in PBS). Incubate for 30 minutes at RT. Flick out and add diluted antibody (diluted in blocking buffer). Incubate for 1 hour at RT. Flick out and wash three times with PBS. Flick out and add second step reagent. Incubate for 1 hour at RT. Flick out and wash three times with PBS. Image sample.

Product Notices

  1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
  2. 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.
  3. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  4. This antibody has been developed and certified for the bioimaging application. However, a routine bioimaging test is not performed on every lot. Researchers are encouraged to titrate the reagent for optimal performance.
  5. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
  6. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
  7. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  8. Triton is a trademark of the Dow Chemical Company.
  9. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
611624 Rev. 2
Antibody Details
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87/eIF-4E

The eukaryotic translation initiation factor 4E (eIF-4E) is a 25 kDa phosphoprotein that specifically binds to the 7-methylguanosine-containing cap of mRNA. eIF-4E is the rate-limiting component for the initiation of cap-dependent translation by the eIF-4E translation initiation complex. This complex promotes the unwinding of secondary structure at the 5' untranslated region of mRNA, which is necessary to expose and locate the AUG-initiation codon. Phosphorylation of eIF-4E on Ser-209 occurs after serum treatment in CHO cells, and may regulate its function. Overexpression of eIF-4E can lead to increased cell proliferation, transformation, and tumorigenesis in nude mice. The overexpression of a Ala-53 variant of eIF-4E cannot evoke these changes, suggesting that Ser-53 on eIF-4E participates in the transfer of mRNA to the 48S initiation complexes. In cooperation with nuclear oncogenes such as c-myc or E1A, eIF-4E transforms primary cells. Other studies have demonstrated that overexpression of eIF-4E causes activation of Ras and leads to a transformed phenotype. Subsequent overexpression of GAP then causes reversion of this phenotype. The mechanism by which eIF-4E plays a role in transformation is not clear, but it is postulated that high levels of eIF-4E may lead to the translation of mRNAs that are normally translationally repressed.

611624 Rev. 2
Format Details
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FITC
Fluorescein (FITC) 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 518-nm. FITC 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.
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FITC
Blue 488 nm
494 nm
518 nm
611624 Rev.2
Citations & References
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Development References (4)

  1. De Benedetti A, Rhoads RE. Overexpression of eukaryotic protein synthesis initiation factor 4E in HeLa cells results in aberrant growth and morphology. Proc Natl Acad Sci U S A. 1990; 87(21):8212-8216. (Biology). View Reference
  2. Lejbkowicz F, Goyer C, Darveau A, Neron S, Lemieux R, Sonenberg N. A fraction of the mRNA 5' cap-binding protein, eukaryotic initiation factor 4E, localizes to the nucleus. Proc Natl Acad Sci U S A. 1992; 89(20):9612-9616. (Biology). View Reference
  3. Rhoads RE. Regulation of eukaryotic protein synthesis by initiation factors. J Biol Chem. 1993; 268(5):3017-3020. (Biology). View Reference
  4. Topisirovic I, Capili AD, Borden KL. Gamma interferon and cadmium treatments modulate eukaryotic initiation factor 4E-dependent mRNA transport of cyclin D1 in a PML-dependent manner. Mol Cell Biol. 2002; 22(17):6183-6198. (Biology). View Reference
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611624 Rev. 2

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