Product Pathways - PathScan ELISA
PathScan® Phospho-Akt (Thr308) Sandwich ELISA Kit #7252
|7252S||1 Kit (96 assays)||---||In Stock||---|
|7252||carrier free and custom formulation / quantity||email request|
When ordering five or more kits, please contact us for processing time and pricing at email@example.com.
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|Kit Includes||Volume||Solution Color|
|Akt Ab Coated Microwells||96 tests|
|P-Akt (T308) Detection Ab||11 ml||Green|
|Anti-mouse IgG, HRP-linked Antibody||11 ml||Red|
|TMB Substrate #7004||11 ml||Colorless|
|STOP Solution #7002||11 ml||Colorless|
|Sealing Tape||2 sheets|
|ELISA Wash Buffer (20X)||25 ml||Colorless|
|ELISA Sample Diluent||25 ml||Blue|
|Cell Lysis Buffer (10X) #9803||15 ml||Yellowish|
Note: 12 8-well modules – Each module is designed to break apart for 8 tests.
Storage: Kit should be stored at 4°C with the exception of Cell Lysis Buffer, which is stored at –20°C (packaged separately).
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.
Specificity / Sensitivity
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.
ELISA - Western correlation
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.
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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- Vlahos, C.J. et al. (1994) J Biol Chem 269, 5241-8.
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- 7160 PathScan® Phospho-Akt1 (Ser473) Sandwich ELISA Kit
- 7170 PathScan® Total Akt1 Sandwich ELISA Kit
- 4691 Akt (pan) (C67E7) Rabbit mAb
- 9272 Akt Antibody
- 2965 Phospho-Akt (Thr308) (C31E5E) Rabbit mAb
- 4056 Phospho-Akt (Thr308) (244F9) Rabbit mAb
- 9275 Phospho-Akt (Thr308) Antibody
- 7076 Anti-mouse IgG, HRP-linked Antibody
- 9803 Cell Lysis Buffer (10X)
- 7004 TMB Substrate
- 7002 STOP Solution
For Research Use Only. Not For Use In Diagnostic Procedures.
PathScan® is a trademark of Cell Signaling Technology, Inc.
Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.