Description
Overview
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
SureFire
Biotin-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
How it works
Phospho-AlphaLISA
SureFire
Biotin Free assay principle
The Phospho-AlphaLISA
SureFire
Biotin Free 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 a proprietary CaptSure 3 agent to capture one of the detection antibodies, which is labeled with CaptSure 3 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 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 permits the cells 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.
