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Figure 3. Dose dependence curve of Akt1 kinase activity: DELFIA® data generated using Phospho-eNOS (Ser1177) (C9C3) Rabbit mAb #9570 to detect phosphorylation of substrate peptide (#1133) by Akt1 kinase. In a 50 µl reaction, increasing amounts of Akt1 and 1.5 µM substrate peptide were used per reaction at room temperature for 10 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Figure 5. Staurosporine inhibition of Akt1 kinase activity: DELFIA® data generated using Phospho-eNOS (Ser1177) (C9C3) Rabbit mAb #9570 to detect phosphorylation of Akt1 substrate peptide (#1133) by Akt1 kinase. In a 50 µl reaction, 10 ng Akt1, 1.5 µM substrate peptide, 50 µM ATP and increasing amounts of staurosporine were used per reaction at room temperature for 10 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Figure 4. Peptide concentration dependence of Akt1 kinase activity: DELFIA® data generated using Phospho-eNOS (Ser1177) (C9C3) Rabbit mAb #9570 to detect phosphorylation of substrate peptide (#1133) by Akt1 kinase. In a 50 µl reaction, 10 ng of Akt1 and increasing concentrations of substrate peptide were used per reaction at room temperature for 10 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Figure 2. Time course of Akt1 kinase activity: DELFIA® data generated using Phospho-eNOS (Ser1177) (C9C3) Rabbit mAb #9570 to detect phosphorylation of Akt1 substrate peptide (#1133) by Akt1 kinase. In a 50 µl reaction, 10 ng Akt1 and 1.5 µM substrate peptide were used per reaction. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Figure 1. Akt1 kinase activity was measured in a radiometric assay using the following reaction conditions: 5 mM MOPS, pH 7.2, 2.5 mM β-glycerophosphate, 1 mM EGTA, 0.4 mM EDTA, 5 mM MgCl2, 0.05 mM DTT, 50 μM ATP, Substrate: Akt-substrate peptide 200 ng/μL, and variable amount of recombinant Akt1.
Product Information
The kit provides a means of performing kinase activity assays with recombinant human Akt1 kinase. It includes active Akt1 kinase (supplied as a GST fusion protein), a biotinylated peptide substrate and a Phospho-serine antibody for detection of the phosphorylated form of the substrate peptide.
| MW (kDa) | Peptide substrate, Biotin-peptide: 2,431 Daltons. GST-Akt1 Kinase: 74 |
| Molecular Formula | Peptide substrate, Biotin-peptide: 2,431 Daltons. GST-Akt1 Kinase: 85 kDa. |
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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