Figure 1: Treatment of NIH/3T3 cells with PDGF stimulates phosphorylation of Akt at Thr308, detected by PathScan® Phospho-Akt (Thr308) Sandwich ELISA Kit #7252, but does not affect the level of total Akt detected by PathScan® Total Akt1 Sandwich ELISA Kit #7170. OD 450 nm readings are shown in the top figure, while the corresponding Western blots using Phospho-Akt (Thr308) Antibody #9275 (right panel) or Akt Antibody #9272 (left panel), are shown in the bottom figure.
Figure 2: The relationship between protein concentration of lysates from untreated and PDGF-treated NIH/3T3 cells and kit assay optical density readings is shown. After starvation, NIH/3T3 cells (85% confluence) were treated with PDGF (50 ng/ml) for 10 min at 37°C, and then lysed.
|Product Includes||Volume||Solution Color|
|Akt Rabbit mAb Coated Microwells||96 tests|
|Phospho-Akt (Thr308) Mouse Detection mAb||1 ea||Green (Lyophilized)|
|Anti-mouse IgG, HRP-linked Antibody (ELISA Formulated)||1 ea||Red (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-Akt (Thr308) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects phospho-Akt (Thr308) protein. An Akt Antibody has been coated onto the microwells. After incubation with cell lysates, both phospho- and nonphospho-Akt proteins are captured by the coated antibody. Following extensive washing, Phospho-Akt (Thr308) Mouse mAb is added to detect the captured phospho-Akt protein. Anti-mouse IgG, HRP-linked Antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of optical density for this developed color is proportional to the quantity of phospho-Akt (Thr308) protein.
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-Akt (Thr308) Sandwich ELISA Kit #7252 detects endogenous levels of phospho-Akt (Thr308) protein. As shown in Figure 1, using Phospho-Akt (Thr308) ELISA Kit #7252, a significant induction of Phospho-Akt (Thr308) is detected in NIH/3T3 cells treated with PDGF. However, levels of total Akt (phospho and nonphospho) detected by PathScan® Total Akt Sandwich ELISA Kit #7170, remain unchanged (Figure 1). Phospho-Akt (Thr308) in Jurkat cells is also detected by this ELISA kit #7252. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other 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).
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