Product Pathways - PI3K / Akt Signaling
Phospho-Akt Pathway Antibody Sampler Kit #9916
|Kit Includes||Quantity||Applications||Reactivity||MW (kDa)||Isotype|
|Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb #4060||40 µl||W IP IHC-P IHC-F IF-IC F||H M R Hm Mk Dm Z B (C) (X) (Dg) (Pg)||60||Rabbit IgG|
|Phospho-Akt (Thr308) (C31E5E) Rabbit mAb #2965||40 µl||W IF-IC F||H M R Hm Mk||60||Rabbit IgG|
|Akt (pan) (C67E7) Rabbit mAb #4691||40 µl||W IP IHC-P IF-IC F||H M R Mk Dm (Pg)||60||Rabbit IgG|
|Phospho-c-Raf (Ser259) Antibody #9421||40 µl||W IP||H M R X (C)||74||Rabbit|
|Phospho-GSK-3β (Ser9) (D85E12) XP® Rabbit mAb #5558||40 µl||W IP IF-IC F||H M R Hm||46||Rabbit IgG|
|Phospho-PTEN (Ser380) Antibody #9551||40 µl||W IP||H M R (C)||54||Rabbit|
|Phospho-PDK1 (Ser241) (C49H2) Rabbit mAb #3438||40 µl||W IP||H M R||58 to 68||Rabbit IgG|
|Anti-rabbit IgG, HRP-linked Antibody #7074||100 µl||Goat|
|LY294002 #9901||0.3 milligrams||307.34|
Reactivity Key: H=Human M=Mouse R=Rat Hm=Hamster Mk=Monkey C=Chicken Dm=D. melanogaster X=Xenopus Z=Zebrafish B=Bovine Dg=Dog Pg=Pig
Species enclosed in parentheses are predicted to react based on 100% sequence homology.
Specificity / Sensitivity
Each phospho-specific antibody recognizes the phosphorylated form of its target. Akt Antibody recognizes total Akt protein, independent of its phosphorylation state.
Western blot analysis of extracts from NIH/3T3 and Jurkat cells, untreated, PDGF-treated or LY294002-treated as indicated, using Phospho-Akt (Thr308) (C31E5) Rabbit mAb #2965 (upper) or Akt (pan) (C67E7) Rabbit mAb #4691 (lower).
Western blot analysis of extracts from PC3 cells, untreated or λ phosphatase-treated, using Phospho-PDK1 (Ser241) (C49H2) Rabbit mAb #3438 (upper), PDK1 Antibody #3062 (middle) or Akt Antibody #9272 (lower).
Western blot analysis of extracts from PC3 cells, untreated or LY294002/wortmannin-treated, and NIH/3T3 cells, serum- starved or PDGF-treated, using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb #4060.
Western blot analysis of extracts from GSK-3α (-/-) (lanes 1,2) and GSK-3β (-/-) (lanes 3,4) mouse embryonic fibroblast (MEF) cells, λ phosphatase or PDGF-treated, using Phospho-GSK-3β (Ser9) (D85E12) XP® Rabbit mAb #5558 (upper) and α/β-Tubulin Antibody #2148 (lower). (MEF wild type, GSK-3α (-/-) and GSK-3β (-/-) cells were kindly provided by Dr. Jim Woodgett, University of Toronto, Canada).
Western blot analysis of extracts from wild type (lanes 1,2), GSK-3α (-/-) (lanes 3,4) and GSK-3β (-/-) (lanes 5,6) mouse embryonic fibroblast cells (MEF), untreated or PDGF treated, using Phospho-GSK-3β (Ser9) (5B3) Rabbit mAb #9323 (upper) and GSK-3α/β Antibody (lower). (MEF wild type, GSK-3α (-/-) and GSK-3β (-/-) cells were kindly provided by Dr. Jim Woodgett, University of Toronto, Canada).
Western blot analysis of extracts from HeLa cells, untreated or TPA-treated, using Phospho-Raf (Ser259) Antibody #9421 (upper), or a total c-Raf antibody (lower).
Western blot analysis of various cell lines with Phospho-PTEN (Ser380) Antibody #9551 (upper) or PTEN Antibody #9552 (lower). The phospho-specificity of the antibody was characterized by treating the membrane without (-) or with (+) calf intestinal alkaline phosphatase (CIP) after Western transfer.
The Phospho-Akt Pathway Antibody Sampler Kit provides an economical means to evaluate the activation status of the Akt signaling pathway, including PTEN and phosphorylated Akt, GSK-3beta, c-Raf and PDK1. The kit includes enough primary and secondary antibodies to perform four Western blot experiments, as well as a specific inhibitor of PI3 kinase (LY294002).
Source / Purification
Antibodies are produced by immunizing rabbits with synthetic phosphopeptides corresponding to residues surrounding Ser473 or Thr308 of mouse Akt, Ser9 of human GSK-3β, Ser259 of human c-Raf, Ser380 of human PTEN or Ser241 of human PDK1. Polyclonal antibodies are purified by protein A and peptide affinity chromatography.
Akt, also referred to as PKB or Rac, plays a critical role in controlling survival and apoptosis (1-3). This protein kinase is activated by insulin and various growth and survival factors to function in a wortmannin-sensitive pathway involving PI3 kinase (2,3). Akt is activated by phospholipid binding and activation loop phosphorylation at Thr308 by PDK1 (4) and by phosphorylation within the carboxy terminus at Ser473. The previously elusive PDK2 responsible for phosphorylation of Akt at Ser473 has been identified as mammalian target of rapamycin (mTOR) in a rapamycin-insensitive complex with rictor and Sin1 (5,6). Akt promotes cell survival by inhibiting apoptosis through phosphorylation and inactivation of several targets, including Bad (7), forkhead transcription factors (8), c-Raf (9), and caspase-9. PTEN phosphatase is a major negative regulator of the PI3 kinase/Akt signaling pathway (10). LY294002 is a specific PI3 kinase inhibitor (11). Another essential Akt function is the regulation of glycogen synthesis through phosphorylation and inactivation of GSK-3α and β (12,13). Akt may also play a role in insulin stimulation of glucose transport (12). In addition to its role in survival and glycogen synthesis, Akt is involved in cell cycle regulation by preventing GSK-3β-mediated phosphorylation and degradation of cyclin D1 (14) and by negatively regulating the cyclin dependent kinase inhibitors p27 Kip1 (15) and p21 Waf1/Cip1 (16). Akt also plays a critical role in cell growth by directly phosphorylating mTOR in a rapamycin-sensitive complex containing raptor (17). More importantly, Akt phosphorylates and inactivates tuberin (TSC2), an inhibitor of mTOR within the mTOR-raptor complex (18,19).
- Franke, T.F. et al. (1997) Cell 88, 435-7.
- Burgering, B.M. and Coffer, P.J. (1995) Nature 376, 599-602.
- Franke, T.F. et al. (1995) Cell 81, 727-36.
- Alessi, D.R. et al. (1996) EMBO J 15, 6541-51.
- Sarbassov, D.D. et al. (2005) Science 307, 1098-101.
- Jacinto, E. et al. (2006) Cell 127, 125-37.
- Cardone, M.H. et al. (1998) Science 282, 1318-21.
- Brunet, A. et al. (1999) Cell 96, 857-68.
- Zimmermann, S. and Moelling, K. (1999) Science 286, 1741-4.
- Cantley, L.C. and Neel, B.G. (1999) Proc Natl Acad Sci USA 96, 4240-5.
- Vlahos, C.J. et al. (1994) J Biol Chem 269, 5241-8.
- Hajduch, E. et al. (2001) FEBS Lett 492, 199-203.
- Cross, D.A. et al. (1995) Nature 378, 785-9.
- Diehl, J.A. et al. (1998) Genes Dev 12, 3499-511.
- Gesbert, F. et al. (2000) J Biol Chem 275, 39223-30.
- Zhou, B.P. et al. (2001) Nat Cell Biol 3, 245-52.
- Navé, B.T. et al. (1999) Biochem J 344 Pt 2, 427-31.
- Inoki, K. et al. (2002) Nat Cell Biol 4, 648-57.
- Manning, B.D. et al. (2002) Mol Cell 10, 151-62.
- Crowder, R.J. and Freeman, R.S. (2000) J Biol Chem 275, 34266-71. Applications: Western Blotting
- Guo, M. et al. (2000) Biochem Pharmacol 60, 635-42. Applications: PI3K inhibition
Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!
* Product-specific protocol.
- 9316 Phospho-GSK-3α (Ser21) (36E9) Rabbit mAb
- 9327 Phospho-GSK-3α/β (Ser21/9) (37F11) Rabbit mAb (GSK-3α Preferred)
- 9940 Akt Isoform Antibody Sampler Kit
- 9292 Bad Antibody
- 9901 LY294002
- 9951 Wortmannin
- 7074 Anti-rabbit IgG, HRP-linked Antibody
- 7071 Phototope®-HRP Western Blot Detection System, Anti-rabbit IgG, HRP-linked Antibody
- 7720 Prestained Protein Marker, Broad Range (Premixed Format)
- 7727 Biotinylated Protein Ladder Detection Pack
- 7003 20X LumiGLO® Reagent and 20X Peroxide
Selected rabbit monoclonal antibodies are produced under license (granting certain rights including those under U. S. Patent No. 5,675,063 and/or U.S.S.N. 11/476,277) from Epitomics, Inc.
For Research Use Only. Not For Use In Diagnostic Procedures.