Figure 1: Treatment of A-673 cells with insulin stimulates phosphorylation of Akt2 at Ser474, detected by PathScan® Phospho-Akt2 (Ser474) Sandwich ELISA Kit #7048, but does not affect levels of total Akt2 protein detected by PathScan® Total Akt2 Sandwich ELISA Kit #7046. The absorbance readings at 450 nm are shown in the top figure, while the corresponding western blots using Akt2 (5B5) Rabbit mAb #2964 (left panel) and Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb #4060 (right panel) are shown in the bottom figure.
Figure 2: Demonstration of phospho-Akt2 (Ser474) sandwich ELISA kit specificity using recombinant phosphorylated Akt1, Akt2 and Akt3 proteins. Phospho-Akt1 (Ser473) is detected by PathScan® Phospho-Akt1 (Ser473) Sandwich ELISA Kit #7160 while PathScan® Phospho-Akt2 (Ser474) Sandwich ELISA Kit #7048 measures levels of phospho-Akt2 (Ser474). Recombinant phosphorylated Akt protein (1.0 ng per microwell) is assayed using both ELISA kits.
Figure 3: The relationship between protein concentration of lysates from untreated and insulin-treated A-673 cells and the absorbance at 450 nm is shown. After starvation, A-673 cells (75% confluence) were treated with insulin (100 nM) for 10-20 min at 37ºC and then lysed.
|7048C||1 Kit (96 assays)|
|Product Includes||Volume||Solution Color|
|Phospho-Akt (Ser473) Rabbit mAb Coated Microwells||96 tests|
|Akt2 (L53E8) Mouse Detection mAb||1 ea||Green (Lyophilized)|
|Detection Antibody Diluent||11 ml||Green|
|HRP Diluent||11 ml||Red|
|TMB Substrate 7004||11 ml|
|STOP Solution 7002||11 ml|
|Sealing Tape||2 ea|
|ELISA Wash Buffer (20X) 9801||25 ml|
|ELISA Sample Diluent||25 ml||Blue|
|Cell Lysis Buffer (10X) 9803||15 ml|
CST's PathScan® Phospho-Akt2 (Ser474) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Akt2 protein when phosphorylated at Ser474. A phospho-Akt rabbit antibody has been coated on the microwells. After incubation with cell lysates, phospho-Akt protein is captured by the coated antibody. Following extensive washing, Akt2 mouse mAb is added to detect captured Akt2 protein. HRP-linked anti-mouse IgG is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of Akt2 phosphorylated at Ser474.
Antibodies in kit are custom formulations specific to kit.
NOTE: Prepare solutions with purified water.
*NOTE: Some PathScan® ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
posted November 2013
Protocol Id: 204
CST's PathScan® Phospho-Akt2 (Ser474) Sandwich ELISA Kit detects endogenous levels of Akt2 protein phosphorylated at Ser474 in human (A-673) cells, as shown in Figure 1. Phospho-Akt2 kit specificity is demonstrated in Figure 2, while the kit sensitivity is shown in Figure 3. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.Species Reactivity:
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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PathScan is a trademark of Cell Signaling Technology, Inc.