Revision 1
#9955
Store at -20C
4E-BP Antibody Sampler Kit
1 Kit
(6 x 20 microliters)
Orders:
877-616-CELL (2355)
Support:
877-678-TECH (8324)
3 Trask Lane | Danvers | Massachusetts | 01923 | USA
For Research Use Only. Not for Use in Diagnostic Procedures.
| Product Includes | Product # | Quantity | Mol. Wt | Isotype/Source |
|---|---|---|---|---|
| Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb | 2855 | 20 µl | 15 to 20 kDa | Rabbit IgG |
| Non-phospho-4E-BP1 (Thr46) (87D12) Rabbit mAb | 4923 | 20 µl | 15-20 kDa | Rabbit IgG |
| Phospho-4E-BP1 (Ser65) Antibody | 9451 | 20 µl | 15 to 20 kDa | Rabbit |
| 4E-BP1 (53H11) Rabbit mAb | 9644 | 20 µl | 15-20 kDa | Rabbit IgG |
| 4E-BP2 Antibody | 2845 | 20 µl | 15 to 20 kDa | Rabbit |
| Phospho-4E-BP1 (Thr70) Antibody | 9455 | 20 µl | 15 to 20 kDa | Rabbit |
| Anti-rabbit IgG, HRP-linked Antibody | 7074 | 100 µl | Goat |
Please visit cellsignal.com for individual component applications, species cross-reactivity, dilutions, protocols, and additional product information.
Description
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 two Western blots with each antibody.
Storage
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
Background
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).
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).
Background References
- 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.
Trademarks and Patents
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Orders: 877-616-CELL (2355) • [email protected] • Support: 877-678-TECH (8324) • [email protected] •
Web:
cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.
Revision 1
Western blot analysis of extracts from A673 cells, untreated or nocodazole-treated (100 ng/ml, 16 hrs), using 4E-BP2 Antibody (upper) or 4E-BP1 Antibody #9452 (lower). Extracts were treated with lambda phosphatase NEB#P0753 (10,000 U/ml for 1 hour) to dephosphorylate both proteins.
Western blot analysis of extracts from 293T 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 deprivation. Amino acids were replenished for 1 hour. Cells were then either untreated (-) or treated with 100 nM insulin (+) for 30 minutes.
Western blot analysis of bacterially expressed GST-4E-BP1 and of extracts from NIH/3T3 cells, using 4E-BP2 Antibody and 4E-BP1 Antibody #9452.
Orders: 877-616-CELL (2355) • [email protected] • Support: 877-678-TECH (8324) • [email protected] •
Web:
cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.
Revision 1
Immunohistochemical analysis of paraffin-embedded human colon carcinoma using Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human colon carcinoma, showing cytoplasmic and nuclear localization, using 4E-BP2 Antibody.
Immunohistochemical analysis of paraffin-embedded human lymphoma using Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb.
Orders: 877-616-CELL (2355) • [email protected] • Support: 877-678-TECH (8324) • [email protected] •
Web:
cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.
Revision 1
Immunohistochemical analysis of paraffin-embedded human lung carcinoma, using 4E-BP2 Antibody.
Immunohistochemical analysis of paraffin-embedded LNCaP cells, untreated (left) or LY294002-treated (right), using Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb on SignalSlide (TM) Phospho-Akt (Ser473) IHC Controls #8101.
Immunohistochemical analysis of paraffin-embedded human follicular carcinoma (thyroid), using 4E-BP2 Antibody.
Orders: 877-616-CELL (2355) • [email protected] • Support: 877-678-TECH (8324) • [email protected] •
Web:
cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.
Revision 1
Immunohistochemical analysis of paraffin-embedded human colon carcinoma using Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb in the presence of control peptide (left) or Phospho-4E-BP1 (Thr37/46) Blocking Peptide #1052 (right).
Confocal immunofluorescent analysis of 293 cells, expressing either non-targeting shRNA (top) or shRNA targeting 4E-BP1/2 (bottom), using Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb (green). To confirm phospho-specificity, cells were treated with an inhibitor cocktail consisting of LY294002 #9901, U0126 #9903, and Rapamycin #9904 (50 μM; 10 μm; 10 nM; 2 hr) (left), stimulated with insulin (100 nM, 30 min; middle), or processed with λ-phosphatase following insulin stimulation (right). Red = Propidium Iodide (PI)/RNase Staining Solution (#4087).
Flow cytometric analysis of Jurkat cells, untreated (green) or treated with LY294002 #9901, Wortmannin #9951, and U0126 #9903 (50 μM, 1 μM, and 10 μM, 2 hr; blue) using Phospho-4E-BP1 (Thr36/46) (236B4) Rabbit mAb (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
Orders: 877-616-CELL (2355) • [email protected] • Support: 877-678-TECH (8324) • [email protected] •
Web:
cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.
Revision 1
Simple Western™ analysis of lysates (0.1 mg/mL) from 3T3 cells using Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb #2855. The virtual lane view (left) shows the target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
Western blot analysis of extracts from COS cells, treated with λ phosphatase or 20% FBS as indicated, using Non-phospho-4E-BP1 (Thr46) (87D12) Rabbit mAb (upper), Phospho-4E-BP1 (Thr37/46) Antibody #9459 (middle), and 4E-BP1 Antibody #9452 (lower).
After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.
Orders: 877-616-CELL (2355) • [email protected] • Support: 877-678-TECH (8324) • [email protected] •
Web:
cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.
Revision 1
Confocal immunofluorescent analysis of 293 shRNA Scramble cells either serum starved (far left); serum starved and treated with U0126 #9903 (10 μM, 2 hr), LY294002 #9901 (50 μM, 2 hr), and Rapamycin #9904 (10 nM, 2 hr) (center, left); serum starved and treated with l phosphatase (10,000 U/mL, 2 hr) ) (center, right); and 293 shRNA 4E-BP1/2 KO treated with U0126 #9903 (10 μM, 2 hr), LY294002 #9901 (50 μM, 2 hr), and Rapamycin #9904 (10 nM, 2 hr) (far right), using Non-phospho-4E-BP1 (Thr46) (87D12) Rabbit mAb (green). Red = Propidium Iodide (PI)/RNase Staining Solution.
Flow cytometric analysis of HEK-293 cells, treated with Insulin (100 nM, 30 min) (blue) or treated with U0126 #9903 (10 µm, 2 hr), LY294002 #9901 (50 µM, 2 hr), and Rapamycin #9904 (10 µM, 2 hr) (green), using Non-phospho-4E-BP1 (Thr46) (87D12) Rabbit mAb (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines) post-fixation/permeabilization. Anti-rabbit IgG F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody
Flow cytometric analysis of HEK-293 cells treated with insulin (100 nM, 30 min), and then treated post-fixation/permeabilization with λ phosphatase (green) or untreated (blue), using Non-phospho-4E-BP1 (Thr46) (87D12) Rabbit mAb (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
Orders: 877-616-CELL (2355) • [email protected] • Support: 877-678-TECH (8324) • [email protected] •
Web:
cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.
Revision 1
Western blot analysis of extracts from 293 cells using 4E-BP1 Antibody #9644 (lower) and Phospho-4E-BP1 (Ser65) Antibody #9451 (upper). The cells were starved for 24 hours in serum-free medium and underwent a 1 hour amino acid deprivation. Amino acids were replenished for 1 hour. Cells were then either untreated (-) or treated with 100 nM insulin (+) for 30 minutes.
Immunoprecipitation of Phospho-4E-BP1 (Ser65) from MCF-7 cell extracts. Cells were starved for 24 hours in serum-free medium and underwent a 1 hour amino acid deprivation. Amino acids were replenished for 1 hour. Cells were then treated with 100 nM insulin for 30 minutes. Lane 1 is 10% input, lane 2 is Normal Rabbit IgG #2729, and lane 3 is Phospho-4E-BP1 (Ser65) Antibody. Western blot was performed using Phospho-4E-BP1 (Ser65) Antibody #9451.
Western blot analysis of extracts from 293 cells, untreated or treated with 20% FBS for the indicated number of minutes, using Phospho-4E-BP1 (Thr70) Antibody (upper) or 4E-BP1 Antibody #9452 (lower).
Orders: 877-616-CELL (2355) • [email protected] • Support: 877-678-TECH (8324) • [email protected] •
Web:
cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.
Revision 1
Western blot analysis of extracts from control HeLa cells (Lane 1) or HeLa cells with a targeted mutation in the gene encoding 4E-BP1 (Lane 2) using 4E-BP1 (53H11) Rabbit mAb (upper) or β-actin (13E5) Rabbit mAb #4970 (lower). The change in 4E-BP1 molecular weight in the mutated HeLa cells confirms the specificity of the antibody for 4E-BP1.
Western blot analysis of extracts from various cell lines using 4E-BP1 (53H11) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human breast carcinoma using 4E-BP1 (53H11) Rabbit mAb in the presence of control peptide (left) or 4E-BP1 blocking peptide #1053 (right).
Orders: 877-616-CELL (2355) • [email protected] • Support: 877-678-TECH (8324) • [email protected] •
Web:
cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.
Revision 1
Immunohistochemical analysis of paraffin-embedded human hepatocellular carcinoma using 4E-BP1 (53H11) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human lung carcinoma using 4E-BP1 (53H11) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded mouse spleen, 4E-BP1/2 wild type (left) or 4E-BP1 knockout (right), using 4E-BP1 (53H11) Rabbit mAb. 4E-BP wild type and knockout tissues kindly provided by Dr. Nahum Sonenberg, McGill University.
Orders: 877-616-CELL (2355) • [email protected] • Support: 877-678-TECH (8324) • [email protected] •
Web:
cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.
Revision 1
Confocal immunofluorescent analysis of HeLa cells using 4E-BP1 (53H11) Rabbit mAb (green). Actin filaments have been labeled with Alexa Fluor® 555 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Confocal immunofluorescent analysis of 293 cells, expressing either nontargeting shRNA (left) or shRNA targeting 4E-BP1/2 (right), using 4E-BP1 (53H11) Rabbit mAb #9644 (green). Red = Propidium Iodide (PI)/RNase Staining Solution #4084.
Flow cytometric analysis of 293 cells, transfected with control shRNA (green) or 4E-BP1-specific shRNA (blue) using 4E-BP1 (53H11) Rabbit mAb (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')₂ Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
Orders: 877-616-CELL (2355) • [email protected] • Support: 877-678-TECH (8324) • [email protected] •
Web:
cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.
Revision 1
Simple Western™ analysis of lysates (0.1 mg/mL) from HepG2 cells using 4E-BP1 (53H11) Rabbit mAb #9644. The virtual lane view (left) shows the target band (as indicated) at 1:50 and 1:250 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:50 (blue line) and 1:250 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 2-40 kDa separation module.
Orders: 877-616-CELL (2355) • [email protected] • Support: 877-678-TECH (8324) • [email protected] •
Web:
cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.