Description
AKT, also known as Protein Kinase B (PKB), is a serine-threonine kinase that regulates critical cellular processes, including survival and apoptosis. There are three isoforms-AKT1, AKT2, and AKT3-encoded by distinct genes (PKBα, PKBβ, and PKBγ). AKT is activated through phosphorylation at threonine 308 by PDK1 and at serine 473 by mTORC2, following binding of its PH domain to phosphoinositides. Dysregulation of AKT isoforms is linked to various diseases: AKT1 is often amplified in breast and gastric cancers, AKT2 in pancreatic cancer and type 2 diabetes due to its role in glucose uptake, and AKT3 in melanoma and aggressive breast tumors.
The AlphaLISA SureFireBiotin-Free Human and Mouse Phospho-AKT1/2/3 (Ser473) Detection Kit is a sandwich immunoassay for the quantitative detection of phospho-AKT1/2/3 in cellular lysates, using Alpha Technology.
Formats:
The HV (high volume) kit contains reagents to run 100 wells in 96-well format, using a 60 μL reaction volume.
The 500-point kit contains enough reagents to run 500 wells in 384-well format, using a 20 μL reaction volume.
The 10,000-point kit contains enough reagents to run 10,000 wells in 384-well format, using a 20 μL reaction volume.
The 50,000-point kit contains enough reagents to run 50,000 wells in 384-well format, using a 20 μL reaction volume.
AlphaLISA SureFire Biotin Free kits are compatible with:
Cell and tissue lysates
Antibody modulators
Biotherapeutic antibodies
Biotin rich samples
AlphaLISA SureFire Biotin Free kits can be used for:
Cellular kinase assays
Receptor activation studies
High-throughput screening for preclinical studies
Phospho-AlphaLISA SureFire Biotin Free assay principle
The Phospho-AlphaLISA SureFire Biotin Free assay measures a protein target when phosphorylated at a specific residue in a biological sample (e.g. cell lysate).
The assay uses two antibodies which recognize the phospho epitope and a distal epitope on the target 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 a proprietary CaptSure3 agent to capture one of the detection antibodies, which is labeled with CaptSure3 tag. 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 for the generation of a luminescent Alpha signal. The amount of light emission is directly proportional to the quantity of phosphoprotein present in the sample.
Phospho-AlphaLISA SureFire Biotin Free two-plate assay protocol
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 Biotin Free detection reagents. This protocol enables cell viability and confluence to be monitored. In addition, lysates from a single well can be used to measure multiple targets.
Phospho-AlphaLISA SureFire Biotin Free one-plate assay protocol
Detection of Phosphorylated target protein with AlphaLISA SureFire Biotin Free 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 robust AlphaLISA SureFire quality.
Activation of Phospho AKT1/2/3 (Ser473) levels in Insulin treated cells
THP-1 cells were seeded in a 96-well plate (400,000 cells/well) and starved in serum free RPMI 1640 at for 2 hours at 37°C, 5% CO2. Cells were then treated with increasing concentrations of Insulin for 5 minutes.
After treatment, the cells were lysed with the addition of 50 µL of 5X Lysis Buffer for 10 minutes at RT with shaking (350 rpm). AKT1/2/3 Phospho (Ser473) and Total levels were evaluated using respective AlphaLISA SureFire Biotin Free assays. For the detection step, 10 µL of cell lysate (approximately 16,000 cells) were 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 AKT1/2/3 (Ser473) while Total AKT1/2/3 levels remained unchanged.
Decrease of Phospho AKT1/2/3 (Ser473) levels in Wortmannin treated cells
PC3 cells were seeded in a 96-well plate (40,000 cells/well) in complete Ham’s F-12K (Kaighn’s) medium, and incubated overnight at 37°C, 5% CO2. The cells were treated with increasing concentrations of Wortmannin for 2 hours in serum free medium.
After treatment, the cells were lysed 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). AKT1/2/3 Phospho (Ser473) and Total levels were evaluated using respective AlphaLISA SureFire Biotin Free assays. For the detection step, 10 µL of cell lysate (approximately 4,000 cells) were 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 AKT1/2/3 (Ser473) while Total AKT1/2/3 levels remained unchanged.
