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Primary Antibodies
Akt/PKB [pT308] Antibody
Background
Akt, also known as Protein Kinase B (PKB) or RAC-α, is a 60 kDa serine/threonine kinase that plays an important role in multiple biological responses including metabolism, cell survival and growth by phosphorylating many proteins including GSK-3β, caspase 9, BAD, and the Forkhead Transcription Factor. Akt is activated by PI3K, which in turn can interact with proteins like FAK (when FAK is phosphorylated at tyrosine 397), thereby linking activation to the cytoskeleton. Akt is phosphorylated on threonine 308 by PDK1 and on serine 473 by PDK2. Phosphorylation at threonine 308 is necessary for phosphorylation of serine 473, which is required for full activation of Akt.
![Akt/PKB [pT308] Antibody](http://www.prosci-inc.com/shop/images/XBP-4034.gif)
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Description
Extracts prepared from HEK293 cells co-expressing RasV12 and RacL61 were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were left untreated (1-5) or treated with lambda (Λ) phosphatase (6), blocked with a 5% BSA-TBST buffer overnight at 4˚C, then incubated with Akt/PKB [pT308] antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 5, 6), the non-phosphorylated peptide corresponding to the immunogen (2), a generic phosphothreonine-containing peptide (3), or, the phosphopeptide immunogen (4). After washing, membranes were incubated with goat F(ab’)2 anti-rabbit IgG HRP-conjugate and bands were detected using the Pierce SuperSignal™ method.
The data show that only the phosphopeptide corresponding to Akt/PKB [pT308] completely blocks the antibody signal, demonstrating the specificity of the antibody.
The data also show that phosphatase stripping eliminates the signal, verifying that the antibody is phospho-specific.
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Source
Akt/PKB antibody was produced against a chemically synthesized phosphopeptide derived from the region of human Akt1 that contains threonine 308. The sequence is conserved among multiple species including mouse and rat Akt1 and Akt2.
Purification
Akt/PKB antibody was purified from rabbit serum by sequential epitope-specific chromatography. The antibody has been negatively preadsorbed using a non-phosphopeptide corresponding to the site of phosphorylation to remove antibody that is reactive with non-phosphorylated Akt protein. The final product is generated by affinity chromatography using an Akt-derived peptide that is phosphorylated at threonine 308.
Clonality / Clone
This is a polyclonal antibody.
Host
Akt/PKB [pT308] antibody was raised in rabbit.
Please use anti-rabbit secondary antibodies.
Specificity
Human and mouse Akt1. Akt1 from other species (100% homologous), Akt2 (100% homologous), and Akt3 (92% homologous) have not been tested, but are expected to react.
Application
For Western blotting applications, we recommend using the antibody at a 1:1000 dilution.
Tested Application
WB
Buffer
Rabbit polyclonal immunoglobulin in Dulbecco’s phosphate buffered saline (without Mg2+ and Ca2+), pH 7.3 (+/- 0.1), 50% glycerol, with 1.0 mg/mL BSA (IgG, protease free) as a carrier.
Storage
Store at -20˚C. We recommend a brief centrifugation before opening to settle vial contents. Then, apportion into working aliquots and store at -20˚C. For shipment or short-term storage (up to one week), 2-8˚C is sufficient.
Positive Control
- 3T3-L1 pre-adipocytes +/- PDGF treatment; HEK293 cells coexperessing RasV12 and RacL61.
Species Reactivity
H, M
Formulation
Rabbit polyclonal immunoglobulin in Dulbecco’s phosphate buffered saline (without Mg2+ and Ca2+), pH 7.3 (+/- 0.1), 50% glycerol, with 1.0 mg/mL BSA (IgG, protease free) as a carrier.
Short Description
Akt/PKB [pT308]
This product belongs to the following categories:
References- Persad, S., et al. (2001) Regulation of protein kinase B/Akt-threonine-308 phosphorylation by integrin linked kinase (ILK): Critical roles for kinase activity and amino acids arginine-211 and threonine-343. J. Biol. Chem. 276(29):27462-27469
- Datta, S.R., et al. (1997) Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell 91(2):231-241.
- Beffert, U., et al. (2002) Reelin-mediated signaling locally regulates protein kinase B/Akt and glycogen synthase kinase 3beta. J. Biol. Chem. 277(51):49958-49964.
- Franke, T.F., et al. (1997) PI3K: downstream AKTion blocks apoptosis. Cell 88(4):435-437.
Datasheet 0801-W
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