REACTIVITY | SENSITIVITY | MW (kDa) | Isotype |
---|---|---|---|
H M R Hm Mk Dm Z B | Endogenous | 60 | Rabbit IgG |
Western blot analysis of extracts of Jurkat cells, treated with either Calyculin A #9902 or LY294002 #9901, using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb (Biotinylated) #5012 and detected with Streptavidin-HRP #3999.
Learn more about how we get our imagesFlow cytometric analysis of Jurkat cells, untreated (green) or treated with LY294002 #9901, Wortmannin #9951, and U0126 #9903 treated (blue), using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb (Biotinylated) compared to XP® Rabbit (DA1E) mAb IgG Isotype Control (Biotinylated) #4096 (red).
Learn more about how we get our imagesFor western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Loading of prestained molecular weight markers (#13953, 10 µl/lane) to verify electrotransfer and biotinylated protein ladder (#7727, 10 µl/lane) to determine molecular weights are recommended.
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
Do not add Anti-biotin, HRP-linked Antibody for detection of biotinylated protein markers. There is no need. The Streptavidin-HRP will also visualize the biotinylated markers.
* Avoid repeated exposure to skin.
posted June 2005
revised November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised June 2016
Protocol Id: 266
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
NOTE: This step is critical for many CST antibodies.
NOTE: Account for isotype matched controls for monoclonal antibodies or species matched IgG for polyclonal antibodies. Count cells using a hemocytometer or alternative method.
posted July 2009
revised September 2013
Protocol Id: 160
Application | Dilutions |
---|---|
Western Blotting | 1:1000 |
Flow Cytometry | 1:400 |
Supplied in 136 mM NaCl, 2.6 mM KCI, 12 mM sodium phosphate (pH 7.4) dibasic, 2 mg/ml BSA, and 50% glycerol. Store at –20°C. Do not aliquot the antibodies.
Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb detects endogenous levels of Akt only when phosphorylated at Ser473.
Human, Mouse, Rat, Hamster, Monkey, D. melanogaster, Zebrafish, Bovine
Chicken, Xenopus, Dog, Pig
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues around Ser473 of human Akt.
This Cell Signaling Technology (CST) antibody is conjugated to biotin under optimal conditions. The unconjugated Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb #4060 reacts with human, mouse, rat, Drosophila melanogaster, hamster, bovine and zebrafish phospho-Akt (Ser473) protein. CST expects that Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb (Biotinylated) will also recognize phospho-Akt (Ser473) in these species.
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).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. XP is a registered trademark of Cell Signaling Technology, Inc.
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Product # | Size | Price |
---|---|---|
5012S | 100 µl (10 western blots) | In Cart |
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