Immunofluorescence analysis of 4EBP1[pT46] was done on 70% confluent log phase HeLa cells. The cells were fixed with 4% paraformaldehyde for 15 minutes; permeabilized with 0.25% Triton™ X-100 for 10 minutes followed by blocking with 5% BSA for 1 hour at room temperature. The cells were incubated with ABfinity™ 4EBP1[pT46] Recombinant Rabbit Oligoclonal Antibody (710323) at a dilution of 2µg/mL in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Flour® 488 Goat Anti-Rabbit IgG Secondary Antibody (A11008) at a dilution of 1:400 for 30 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 594 Phalloidin (A12381). Panel d is a merged image showing nuclear localization of 4EBP1[pT46]. Panel e shows competition with 4EBP1 [pT46] peptide. The images were captured at 20X magnification.
|Tested species reactivity||Human|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Phosopeptide corresponding to amino acids 41–50 of human 4EBP1 [pT46]|
|Contains||0.09% sodium azide|
|Storage Conditions||Maintain refrigerated at 2-8°C for up to 1 month. For long term storage store at -20°C|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||1-3 µg/10^6 cells|
|Immunocytochemistry (ICC)||1 ug/ml|
|Immunofluorescence (IF)||1-3 ug/ml|
|Western Blot (WB)||1-2 ug/ml|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
This antibody is predicted to react with mouse, rat, non-human primate and rabbit based on sequence homology.
ABfinity™ recombinant antibodies are rabbit monoclonal antibodies, unmatched for producing superior results. ABfinity™ antibodies are developed by immunizing animals, screening for functionality, cloning the immunogen-specific antibody genes into high-level mammalian expression vectors, produced on a large scale and purified with Protein A.
ABfinity™ oligoclonal antibodies comprise a selection of multiple different recombinant monoclonal antibodies, providing the best of both worlds—the sensitivity of a polyclonal antibody with the specificity of a monoclonal, all delivered with the consistency only found in a recombinant antibody. While functionally the same as a polyclonal antibody—recognizing multiple epitope sites on the target and producing higher detection sensitivity for low abundance targets when compared with monoclonal antibodies—an oligoclonal antibody has a known mixture of light and heavy chains. This exact population can be produced in every lot, circumventing the biological variability typically associated with polyclonal antibody production.
Intact IgG appears on a non-reducing gel as ~150 kDa band and upon reduction generating a ~25 kDa light chain band and a ~50 kDa heavy chain.
Eukaryotic initiation factor 4E binding protein 1 (4E-BP1), also known as PHAS, is a ~20 kDa member of a family of eIF4E-binding proteins whose binding affinity to eIF4E is regulated by its phosphorylation. It inhibits cap-dependent translation by binding to eIF4E on the same site that overlaps the binding site for eIF4G, preventing its binding to the latter and eventually leading to an increase in mRNA translation. The phosphorylation of 4E-BP1 is critical in determining cell fate by controlling translation initiation and apoptotic potency. 4E-BP1 is hyperphosphorylated in response to several external stimuli including hormones, growth factors, mitogens, cytokines and G-protein–coupled receptors and in response to stress conditions including nutrient deprivation. The phosphorylation of these sites is believed to occur in an orderly fashion where phosphorylation of threonine 37 and 46 by FRAP/mTOR is a priming step for subsequent phosphorylation of 4E-BP1 at the carboxy-terminal sites. Under normoxic conditions, increased VEGF expression, resulting from inhibition of 4E-BP1, contributes to efficient angiogenesis and metastatic brain growth through activated integrin alphav beta3.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.