The AlphaLISA™ SureFire® Ultra™ Human and Mouse Phospho-PRAS40 (Thr246) assay is a sandwich immunoassay for quantitative detection of phospho-PRAS40 in cellular lysates using Alpha Technology.
For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption and disposal requirements under European REACH regulations (EC 1907/2006).
The AlphaLISA™ SureFire® Ultra™ Human and Mouse Phospho-PRAS40 (Thr246) assay is a sandwich immunoassay for quantitative detection of phospho-PRAS40 in cellular lysates using Alpha Technology.
For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption and disposal requirements under European REACH regulations (EC 1907/2006).
Proline-rich AKT substrate 40 kDa (PRAS40) is a substrate of AKT kinase and a component of the mechanistic target of rapamycin complex 1 (mTORC1). Phosphorylation of PRAS40 by AKT relieves its inhibitory effect on mTORC1, thereby promoting cell growth and proliferation. Dysregulation of PRAS40 phosphorylation is implicated in various cancers and metabolic disorders, highlighting its role in cell survival and metabolism.
The AlphaLISA SureFire Ultra Human and Mouse Phospho-PRAS40 (Thr246) Detection Kit is a sandwich immunoassay for the quantitative detection of phospho-PRAS40 (Thr246) in cellular lysates, using Alpha Technology.
Formats:
AlphaLISA SureFire Ultra kits are compatible with:
Alpha SureFire Ultra kits can be used for:
Automation Compatible |
Yes
|
---|---|
Brand |
AlphaLISA SureFire Ultra
|
Detection Modality |
Alpha
|
Host Species |
Human
Mouse
|
Shipping Conditions |
Shipped in Blue Ice
|
Target |
PRAS40
|
Therapeutic Area |
Oncology
|
Unit Size |
500 Assay Points
|
The Phospho-AlphaLISA SureFire Ultra assay measures a protein target when phosphorylated at a specific residue.
The assay uses two antibodies which recognize the phospho epitope and a distal epitope on the targeted protein. AlphaLISA assays require two bead types: Acceptor and Donor beads. Acceptor beads are coated with a proprietary CaptSure™ agent to specifically immobilize the assay specific antibody, labeled with a CaptSure™ tag. Donor beads are coated with streptavidin to capture one of the detection antibodies, which is biotinylated. In the presence of phosphorylated protein, the two antibodies bring the Donor and Acceptor beads in close proximity whereby the singlet oxygen transfers energy to excite the Acceptor bead, allowing the generation of a luminescent Alpha signal. The amount of light emission is directly proportional to the quantity of phosphoprotein present in the sample.
The two-plate protocol involves culturing and treating the cells in a 96-well plate before lysis, then transferring lysates into a 384-well OptiPlate™ plate before the addition of Phospho-AlphaLISA SureFire Ultra detection reagents. This protocol permits the cells' viability and confluence to be monitored. In addition, lysates from a single well can be used to measure multiple targets.
Detection of Phosphorylated target protein with AlphaLISA SureFire Ultra reagents can be performed in a single plate used for culturing, treatment, and lysis. No washing steps are required. This HTS designed protocol allows for miniaturization while maintaining AlphaLISA SureFire Ultra quality.
HeLa cells were seeded in a 96-well plate (20,000 cells/well) in complete medium, and incubated over 48 hours at 37°C, 5% CO2. The cells were starved for 3 hours then treated with increasing concentrations of Insulin for 5 minutes.
After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). PRAS40 Phospho (Thr246) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 2,000 cells) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
As expected, Insulin triggered a dose-dependent increase in the levels of Phospho PRAS40 (Thr246) while Total PRAS40 levels remained unchanged.
A549 cells were seeded in a 96-well plate (40,000 cells/well) in complete medium, and incubated overnight at 37°C, 5% CO2. The cells were treated with increasing concentrations of UCN-01 for 1 hour.
After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). PRAS40 Phospho (Thr246) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 4,000 cells) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
As expected, UCN-01 triggered a dose-dependent decrease in the levels of Phospho PRAS40 (Thr246) while Total PRAS40 levels remained unchanged.
A549 cells were seeded in a 96-well plate (40,000 cells/well) in complete medium, and incubated overnight at 37°C, 5% CO2. The cells were treated with increasing concentrations of Staurosporine for 1 hour.
After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). PRAS40 Phospho (Thr246) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 4,000 cells) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
As expected, Staurosporine triggered a dose-dependent decrease in the levels of Phospho (Thr246) PRAS40 while Total PRAS40 levels remained unchanged.
A549 cells were seeded in a 96-well plate (40,000 cells/well) in complete medium, and incubated overnight at 37°C, 5% CO2. The cells were treated with increasing concentrations of Wortmannin for 1 hour.
After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). PRAS40 Phospho (Thr246) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 4,000 cells) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
As expected, Wortmannin triggered a dose-dependent decrease in the levels of Phospho PRAS40 (Thr246) while Total PRAS40 levels remained unchanged.
Are you looking for resources, click on the resource type to explore further.
We are here to answer your questions.