Product Pathways - Protein Translation
4E-BP Antibody Sampler Kit #9955
|9955S||1 Kit (6 x 40 µl)||---||In Stock||---|
|Kit Includes||Quantity||Applications||Reactivity||MW (kDa)||Isotype|
|Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb #2855||40 µl||W, IHC-P, IF-IC, F||H, M, R, Mk, Dm||15 to 20||Rabbit IgG|
|Non-phospho-4E-BP1 (Thr46) (87D12) Rabbit mAb #4923||40 µl||W, F||H, M, R, Mk||15-20||Rabbit IgG|
|Phospho-4E-BP1 (Ser65) Antibody #9451||40 µl||W, IP||H, M, R, Mk||15 to 20||Rabbit|
|4E-BP1 (53H11) Rabbit mAb #9644||40 µl||W, IP, IHC-P, IF-IC, F||H, M, R, Mk||15-20||Rabbit IgG|
|4E-BP2 Antibody #2845||40 µl||W, IP, IHC-P, F||H, M, R, Mk, B||15 to 20||Rabbit|
|Phospho-4E-BP1 (Thr70) Antibody #9455||40 µl||W, IP||H, M, R, Mk||15 to 20||Rabbit|
|Anti-rabbit IgG, HRP-linked Antibody #7074||100 µl||Goat|
Applications Key: W=Western Blotting, IHC-P=Immunohistochemistry (Paraffin), IF-IC=Immunofluorescence (Immunocytochemistry), F=Flow Cytometry, IP=Immunoprecipitation
Reactivity Key: H=Human, M=Mouse, R=Rat, Mk=Monkey, Dm=D. melanogaster, B=Bovine
Western blot analysis of bacterially expressed GST-4E-BP1 and of extracts from NIH/3T3 cells using 4E-BP2 Antibody #2845 and 4E-BP1 Antibody #9452.
Western blot analysis of extracts from 293 cells using 4E-BP1 Antibody #9452 (upper) and Phospho-4E-BP1 (Thr37/46) Antibody #2855 (lower). The cells were starved for 24 hours in serum-free medium and underwent a 1 hour amino acid depravation. Amino acids were then added back for 1 hour, and cells were either untreated (-) or treated with 100 nM insulin (+) for 30 minutes.
Western blot analysis of extracts from COS cells, λ phosphatase-treated or 20% FBS-treated, using Nonphospho-4E-BP1 (Thr46) (87D12) Rabbit mAb #4923 (upper), Phospho-4E-BP1 (Thr37/46) Antibody #9459 (middle), and 4E-BP1 Antibody #9452 (lower).
Western blot analysis of extracts from C6 and MEF cells using Phospho-4E-BP1 (Ser65) Antibody #9451 (upper) or 4E-BP1 (53H11) Rabbit mAb #9644 (lower).
The 4E-BP Antibody Sampler Kit provides an economical means to investigate regulation of cap-dependent translation within the cell. The kit contains primary and secondary antibodies to perform four Western blots with each antibody.
Specificity / Sensitivity
Phospho-4E-BP1 (Thr37/46) Rabbit mAb detects endogenous levels of 4E-BP1 only when phosphorylated at Thr37 and/or Thr46, and may cross-react with 4E-BP2 and 4E-BP3 when phosphorylated at equivalent sites. Nonphospho-4E-BP1 (Thr46) (87D12) Rabbit mAb detects endogenous levels of 4E-BP1 only when dephosphorylated at Thr46. This antibody cross-reacts with 4E-BP2 and 4E-BP3 dephosphorylated at equivalent sites. Phospho-4E-BP1 (Ser65) Antibody detects endogenous levels of 4E-BP1 when phosphorylated at Ser65, and may also recognize 4E-BP1 when phosphorylated at Ser101. Phospho-4E-BP1 (Ser65) (174A9) Rabbit mAb detects endogenous levels of 4E-BP1 when phosphorylated at Ser65. Phospho-4E-BP1 (Thr70) Antibody detects endogenous levels of 4E-BP1 only when phosphorylated at Thr70. 4E-BP1 (53H11) Rabbit mAb detects endogenous levels of total 4E-BP1 protein. 4E-BP2 Antibody detects endogenous levels of total 4E-BP2 independent of phosphorylation and does not cross-react significantly with 4E-BP1.
Source / Purification
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr37 and Thr46 of mouse 4E-BP1, residues surrounding Thr46 of human 4E-BP1, or Ser112 of human 4E-BP1. Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to the residues at the carboxy-terminus of human 4E-BP2 (#2845), or phosphopeptides surrounding mouse Ser65 (#9451) and human Thr70 (#5078) 4E-BP1. Polyclonal antibodies were purified by protein A and peptide affinity chromatography.
Translation repressor protein 4E-BP1 (also known as PHAS-1) inhibits cap-dependent translation by binding to the translation initiation factor eIF4E. Hyperphosphorylation of 4E-BP1 disrupts this interaction and results in activation of cap-dependent translation (1). Both the PI3 kinase/Akt pathway and FRAP/mTOR kinase regulate 4E-BP1 activity (2,3). Multiple 4E-BP1 residues are phosphorylated in vivo (4). While phosphorylation by FRAP/mTOR at Thr37 and Thr46 does not prevent the binding of 4E-BP1 to eIF4E, it is thought to prime 4E-BP1 for subsequent phosphorylation at Ser65 and Thr70 (5).
4E-BP2 and 4E-BP3 share high sequence homology with 4E-BP1, including conservation of the major FRAP/mTOR-dependent phosphorylation sites. Preliminary data suggests that phosphorylation of 4E-BP2 is regulated in a similar manner to that of 4E-BP1, although phosphorylation of this protein has not been as extensively studied (6).
- Pause, A. et al. (1994) Nature 371, 762-7.
- Brunn, G.J. et al. (1997) Science 277, 99-101.
- Gingras, A.C. et al. (1998) Genes Dev 12, 502-13.
- Fadden, P. et al. (1997) J Biol Chem 272, 10240-7.
- Gingras, A.C. et al. (1999) Genes Dev 13, 1422-37.
- Lin, T.A. and Lawrence, J.C. (1996) J. Biol. Chem. 271, 30199-30204.
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- 7003 20X LumiGLO® Reagent and 20X Peroxide
- 7074 Anti-rabbit IgG, HRP-linked Antibody
This product is intended for research purposes only. The product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.
Select rabbit monoclonal antibodies are developed, validated, and produced at CST using in part technology under license (granting certain rights including those under U.S. Patents No. 5,675,063 and in some instances 7,429,487) from Epitomics, Inc.