Immunohistochemical analysis of paraffin-embedded human breast carcinoma, showing cytoplasmic and nuclear localization, using Phospho-Akt (Ser473) (736E11) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human lung carcinoma, using Phospho-Akt (Ser473) (736E11) Rabbit mAb.
Immunohistochemical analysis using Phospho-Akt (Ser473) (736E11) Rabbit mAb on SignalSlide (R) Phospho-Akt (Ser473) IHC Controls #8101 (paraffin-embedded LNCaP cells, untreated (left) or LY294002-treated (right).
Immunohistochemical analysis of paraffin-embedded human prostate carcinoma, using Phospho-Akt (Ser473) (736E11) Rabbit mAb preincubated with an irrelevant peptide (left) or Phospho-Akt (Ser473) Blocking Peptide (#1140) (right).
Immunohistochemical analysis of paraffin-embedded MDA-MB-468 xenograft, using Phospho-Akt (Ser473) (736E11) Rabbit mAb (left) or PTEN (138G6) Rabbit mAb #9559 (right). MDA-MB-468 cells lack PTEN. Note the presence of P-Akt in the PTEN deficient cells.
Immunohistochemical analysis of frozen U-87MG xenograft, showing predominantly cytoplasmic localization using Phospho-Akt (Ser473)(736E11) Rabbit mAb.
This Cell Signaling Technology antibody is conjugated to biotin under optimal conditions. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated
Phospho-Akt (Ser473) (736E11) Rabbit mAb #3787.
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.
NOTE: Do not allow slides to dry at any time during this procedure.
For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; follow with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.
|SignalStain® Boost IHC Detection Reagent (HRP, Rabbit) #8114||SignalStain® Boost IHC Detection Reagent (AP, Rabbit) #18653|
|SignalStain® DAB Substrate Kit #8059||SignalStain® Vibrant Red Alkaline Phosphatase Substrate Kit #76713|
|SignalStain® Vivid Purple Peroxidase Substrate Kit #96632|
NOTE: Use of detection reagents other than those specified in this protocol may require further optimization of the primary antibody to account for the different sensitivities of the detection reagents.
posted June 2005
revised June 2020
Protocol Id: 303
Wash Buffer: 1X Tris Buffered Saline (TBS).
To prepare 1L 1X TBS add 100 ml 10X Tris Buffered Saline (#12498) to 900 ml dH2O, mix.
posted January 2006
revised March 2016
Protocol Id: 330
Phospho-Akt (Ser473) (736E11) Rabbit mAb detects Akt1 only when phosphorylated at serine 473, and Akt2 and Akt3 only when phosphorylated at equivalent sites.
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser473 of mouse Akt.
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).
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