Description
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.
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.
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.
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.
