Based on our homogeneous and robust HTRF assays, phospho-AKT kits are designed for detecting and studying activated AKT when phosphorylated either at Ser 473 or at Thr 308 directly in whole cells. Using streamlined protocols, amenable to low-volume formats, this kit can be used from basic research to High Throughput drug screening.
The 2 assays can be run with cell lysates or using whole cells. Upon activation, AKTis phosphorylated either on Ser473 or on Thr308, and after cell membrane lysis, phospho- AKTcan be detected using the kit reagents. The assays are based on sandwich immunoassays, each involving two monoclonal antibodies: the anti-phospho-AKT antibody labeled with Eu3+-cryptate and the anti-AKT antibody labeled with d2. These antibodies may be pre-mixed and added in a single dispensing step to further streamline the protocol.
Cells are plated, stimulated and next lysed in the same 96-well culture plate. Lysates are then transferred to the assay plate for the detection of phosphorylated AKT by HTRF reagents. This protocol enables cell viability and confluence to be monitored.
Detection of phosphorylated AKT with HTRF reagents is performed in a single plate used for plating, stimulation and lysis. No washing steps are required. This protocol, HTS designed, allows miniaturization while maintaining HTRF quality.
AKT (or protein kinase B) plays a key role in controlling survival and apoptosis. This serine/threonine protein kinase is regulated by insulin and various growth and survival factors, to work in a wortmannin-sensitive pathway involving PI 3 kinase. When the Pleckstrin Homology (PH) domain of AKT binds to phosphoinositides, AKT can be phosphorylated by two different kinases, PDK1 at threonine 308 and mTORC2 (mammalian target of rapamycin) at serine 473, which switch on AKT activation. Besides being a downstream effector of PI3-kinases, AKT may also be activated in a PI 3-kinase-independent manner.
A431 cells were grown in a T175 flask 37°C, 5%Co2, 2days. Stimulation was done with 100nM EGF for 10min. After elimination of cell culture medium, 3ml of supplemented lysis buffer was added and incubated for 45min. Soluble supernatants were collected after 10min centrifuging. Total protein concentration was determined by BCA assay: 20.106 cells lysed under 3ml corresponds to 3.2mg/ml of total proteins. (50.000 cells per well corresponds to 25µg of total proteins) 16µl of serial lysates dilutions were dispensed and analysed side-by-side by WB and HTRF assay. The HTRF assay is 16-fold more sensitive than the western-blot : 200 cells can be detected by using Phospho-AKT (Ser473) while 3125 cells are needed for the Western-blot.
Results obtained on CHO cells plated using 2 cell densities (25,000 - 50,000 cells per well). After being starved for 24 hours, cells were stimulated for 10 minutes with various concentrations of IGF. Phosphorylated AKT on Thr308 was measured after 3.5 hours using the two-plate assay protocol.
Results obtained on HEK cells plated using 3 cell densities (12,500 - 25,000 - 50,000 cells per well). After being starved for 24 hours, cells were stimulated for 10 minutes with various concentrations of IGF. Phosphorylated AKT was measured after 3.5 hours using the two-plate assay protocol.
Cells plated at 50,000 cells per well were starved for 16 hours and then stimulated at room temperature for 3 different incubation times - 3, 7 and 15 min - with various concentrations of growth factor based ligand stimulator.
Stimulation curves shown here were obtained using the two-plate assay protocol of the Cellul'erk assay for the assessment of phosphorylated Erk (fig. A) and the two-plate assay protocol of the Phospho-AKT assay for the study of phosphorylated AKT (fig.B).
Results used with permission of Acorda Therapeutics, 15 Skyline Drive, Hawthorne NY 10532, United States.
| Description | Cat. no | Product insert | MSDS |
|---|---|---|---|
| Phospho AKT (Ser473) Assay kit - 500 tests | 64AKSPEG | ||
| Phospho AKT (Ser473) Assay kit - 10,000 tests | 64AKSPEH | ||
| Phospho AKT (Ser473) Assay kit - 50,000 tests | 64AKSPEI | ||
| Phospho AKT (Thr308) Assay kit - 500 tests | 64AKTPEG | ||
| Phospho AKT (Thr308) Assay kit - 10,000 tests | 64AKTPEH | ||
| Phospho AKT (Thr308) Assay kit - 50,000 tests | 64AKTPEI |
| Description | Cat. no | Product insert | MSDS |
|---|---|---|---|
| Phospho AKT (Ser473) control lysate | 62AKSTDA | - | - |
| Phospho AKT (Thr308) control lysate | 62AKTTDA | - | - |
| Cellular kinase blocking reagent - 2 ml | 64KB1AAC | - | |
| Cellular kinase blocking reagent - 6 ml | 64KB1AAD | - | |
| Cellular kinase lysis buffer - 130 mL | 64KL1FDF | - | |
| Total AKT Assay kit - 500 tests | 64NKTPEG | ||
| Total AKT Assay kit - 10,000 tests | 64NKTPEH | ||
| Total AKT Assay kit - 50,000 tests | 64NKTPEI |
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