Synthetic peptide (the amino acid sequence is considered to be commercially sensitive) (N terminal)
293 cell lysate and human colon carcinoma
This product is a recombinant rabbit monoclonal antibody.
Binding of eIF4EBP1 to eIF4E is reversible and is dependent on the phosphorylation status of eIF4EBP1. Non phosphorylated eIF4EBP1 will bind strongly to eIF4E while, the phosphorylated form will not. Akt, TOR, MAP kinase, S6 kinase, and Cdc2 are known kinases capable of inactivating eIF4EBP1 binding to eIF4E by phosphorylating either threonines 35, 45, 69 or serine 64. Although, not all phosphorylation events equally block the eIF4EBP1-eIF4E interaction
Produced using Abcam's RabMAb® technology. RabMAb® technology is covered by the following U.S. Patents, No. 5, 675, 063 and/or 7, 429, 487.
Storage instructionsShipped at 4°C. Upon delivery aliquot and store at -20°C. Avoid freeze / thaw cycles.
Primary antibody notesBinding of eIF4EBP1 to eIF4E is reversible and is dependent on the phosphorylation status of eIF4EBP1. Non phosphorylated eIF4EBP1 will bind strongly to eIF4E while, the phosphorylated form will not. Akt, TOR, MAP kinase, S6 kinase, and Cdc2 are known kinases capable of inactivating eIF4EBP1 binding to eIF4E by phosphorylating either threonines 35, 45, 69 or serine 64. Although, not all phosphorylation events equally block the eIF4EBP1-eIF4E interaction
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
1/5000 - 1/10000. Detects a band of approximately 17 kDa (predicted molecular weight: 13 kDa).
Use at an assay dependent concentration.
1/50 - 1/100.
ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
Use at an assay dependent concentration.
FunctionRegulates eIF4E activity by preventing its assembly into the eIF4F complex. Mediates the regulation of protein translation by hormones, growth factors and other stimuli that signal through the MAP kinase and mTORC1 pathways.
Sequence similaritiesBelongs to the eIF4E-binding protein family.
Post-translational modificationsPhosphorylated on serine and threonine residues in response to insulin, EGF and PDGF. Phosphorylation at Thr-37, Thr-46, Ser-65 and Thr-70 is regulated by mTORC1. Phosphorylated upon DNA damage, probably by ATM or ATR.
Immunocytochemistry/Immunofluorescence analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) labeling eIF4EBP1 with Purified ab32024 at 1/500 dilution (5 µg/ml). Cells were fixed with 4% PFA and permeabilized with 0.1% tritonX-100. ab150077 Goat anti rabbit IgG(Alexa Fluor® 488) at 1/1000 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI. PBS was used instead of the primary antibody as the negative control.
Western blot - Anti-eIF4EBP1 antibody [Y329] (ab32024)
Predicted band size : 13 kDa
Lane 1: Wild-type HAP1 cell lysate (20 µg) Lane 2: elF4EBP1 knockout HAP1 cell lysate (20 µg) Lane 3: HEK293 cell lysate (20 µg) Lane 4: K562 cell lysate (20 µg) Lanes 1 - 4: Merged signal (red and green). Green - ab32024 observed at 20 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab32024 was shown to specifically react with elF4EBP1 when elF4EBP1 knockout samples were used. Wild-type and elF4EBP1 knockout samples were subjected to SDS-PAGE. ab32024 and ab8245 (loading control to GADPH) were diluted 1/5000 and 1/2000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10000 dilution for 1 h at room temperature before imaging.
Overlay histogram showing HT1080 cells stained with ab32024 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab32024, 1/50 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x106 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in HT1080 cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.
References for Anti-eIF4EBP1 antibody [Y329] (ab32024)
This product has been referenced in:
Zhang W et al. Activation of mTOR in the spinal cord is required for pain hypersensitivity induced by chronic constriction injury in mice. Pharmacol Biochem Behav111:64-70 (2013).
Read more (PubMed: 23948070) »
Silver N et al. Activation of mTOR coincides with autophagy during ligation-induced atrophy in the rat submandibular gland. Cell Death Dis1: (2010).
Read more (PubMed: 20890458) »