Description
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 threonines 36 and 45 (T36/T45) are phosphorylated in resting human peripheral blood mononuclear cells. PI3 kinase inhibitors, such as LY294002 down-regulate the phosphorylation level of 4EBP1 (pT36/pT45). The M31-16 monoclonal antibody recognizes the phosphorylated T36 and T45 of activated human 4EBP1. The orthologous phosphorylation sites in mouse and rat 4EBP1 are T35 and T44.
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.
