Product nameAnti-eIF4EBP1 antibody [Y329]
See all eIF4EBP1 primary antibodies
DescriptionRabbit monoclonal [Y329] to eIF4EBP1
Tested applicationsSuitable for: WB, IHC-P, Flow Cyt, IP, ICC/IFmore details
Species reactivityReacts with: Mouse, Rat, Human
Synthetic peptide within Human eIF4EBP1 aa 1-100 (N terminal). The exact sequence is proprietary.
- 293 cell lysate and human colon carcinoma Flow Cyt: HAP1-WT cells.
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.
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents
This product is a recombinant rabbit monoclonal antibody.
Storage instructionsShipped at 4°C. Upon delivery aliquot and store at -20°C. Avoid freeze / thaw cycles.
Storage bufferpH: 7.20
Preservative: 0.01% Sodium azide
Constituents: 49% PBS, 50% Glycerol, 0.05% BSA
Concentration information loading...
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
Our Abpromise guarantee covers the use of ab32024 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|WB||1/5000 - 1/10000. Detects a band of approximately 17 kDa (predicted molecular weight: 13 kDa).|
|IHC-P||1/100 - 1/250.|
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.
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.
- Information by UniProt
- 4E-BP1 antibody
- 4EBP1 antibody
- 4EBP1_HUMAN antibody
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 HAP1 wildtype (green line) and HAP1-EIF4EBP1 knockout cells (red line) stained with ab32024. The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab32024, 0.1µg/ml) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) presorbed (ab150081) at 1/2000 dilution for 30 min at 22°C.
A rabbit IgG isotype control antibody (ab172730) was used at the same concentration and conditions as the primary antibody (HAP1 wildtype - black line, HAP1-EIF4EBP1 knockout - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).
Acquisition of >5,000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter.
This antibody can also be used in HAP1 cells fixed with 4% formaldehyde (10 min) , permeabilized with 0.1% PBS-Triton X-100 for 15 min under the same conditions.
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.
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.
ab32024, at a 1/100 dilution, staining human eIF4EBP1 in colon carcinoma by Immunohistochemistry, Paraffin embedded tissue
Anti-eIF4EBP1 antibody [Y329] (ab32024) at 1/10000 dilution + 293 cell lysate
Predicted band size: 13 kDa
Observed band size: 15 - 20 kDa why is the actual band size different from the predicted?
This product has been referenced in:
- Li F et al. Expression of 4E-BP1 and phospho-4E-BP1 correlates with the prognosis of patients with clear cell renal carcinoma. Cancer Manag Res 10:1553-1563 (2018). Read more (PubMed: 29942157) »
- Liu Y et al. Effects of bufalin on the mTOR/p70S6K pathway and apoptosis in esophageal squamous cell carcinoma in nude mice. Int J Mol Med 40:357-366 (2017). Read more (PubMed: 28656204) »