AKT3 is a serine/threonine protein kinase and a key member of the PI3K/AKT signaling pathway, playing critical roles in brain development, neuronal survival, and cellular growth regulation. AKT3 is activated by phosphorylation at Thr305 and Ser472 downstream of PI3K, promoting protein synthesis, cell survival, and glucose metabolism while regulating cell size and proliferation. It is particularly important in the central nervous system, where it controls brain size, neuronal differentiation, and synaptic plasticity. Mutations or altered expression of AKT3 are associated with microcephaly, macrocephaly, neurological disorders, and various cancers including melanoma, breast, and prostate carcinomas. In oncology, AKT3 promotes tumor cell survival, invasion, and drug resistance while supporting cancer stem cell maintenance and epithelial-to-mesenchymal transition. Therapeutic targeting of AKT3 through selective inhibitors represents a promising strategy for both neurodevelopmental disorders and aggressive cancer subtypes, particularly those with enhanced metastatic potential.
The AlphaLISA SureFire Ultra Human and Mouse Phospho-AKT3 (Ser472) is a sandwich immunoassay for the quantitative detection of phospho-AKT3 (Ser472) 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 Ultra kits are compatible with:
Cell and tissue lysates
Antibody modulators
Biotherapeutic antibodies
AlphaLISA SureFire Ultra kits can be used for:
Cellular kinase assays
Receptor activation studies
High-throughput screening for preclinical studies
Phospho-AlphaLISA SureFire Ultra assay principle
The Phospho-AlphaLISA SureFire Ultra assay measures a target protein 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 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 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 Ultra 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 Ultra 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 Ultra one-plate assay protocol
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 robust AlphaLISA SureFire Ultra quality.
Activation of AKT3 Phospho (Ser472) in insulin treated cells
SH-SY5Y and NIH/3T3 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 starved for 2 hours and then treated with increasing concentrations of insulin for 30 minutes.
After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). AKT3 Phospho (Ser472) 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, insulin triggered a dose-dependent increase in the levels of AKT3 Phospho (Ser472) while total levels remained unchanged.
AKT3 phosphorylation induced by TIE2 pathway activation
HUVEC cells were seeded in a 96-well plate (20,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO2. The cells were starved for 2 hours and then treated with increasing concentrations of Razuprotafib for 15 minutes.
After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). AKT3 Phospho (Ser472) 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, Razuprotafib triggered a dose-dependent increase in the levels of AKT3 Phospho (Ser472) while total levels remained unchanged.
Inhibition of AKT3 Phospho (Ser472) in HEK293 cells
HEK293 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 for 24 hours with increasing concentrations of Luminespib for 24 hours.
After treatment, the cells were lysed with 200 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). AKT3 Phospho (Ser472) 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, Luminespib triggered a dose-dependent decrease in the levels of AKT3 Phospho (Ser472) while total levels remained unchanged.
HEK293 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 for 2 hours with increasing concentrations of Wortmannin for 2 hours.
After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). AKT3 Phospho (Ser472) 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 AKT3 Phospho (Ser472) while total levels did not change significantly.
Specificity of AKT3 Phospho (Ser472) assay
Specificity of the AKT3 Phospho (Ser472) assay for AKT3 protein was assessed by assaying active recombinant AKT1, AKT2 and AKT3 proteins.
Dilutions of AKT1 (Abcam, ab62279), AKT2 (Abcam, ab60324) and AKT3 (Abcam, ab60324) proteins were prepared in Lysis Buffer at the indicated concentrations. AKT3 Phospho (Ser472) signal was evaluated using the AlphaLISA SureFire Ultra assay. For the detection step, 10 µL of protein was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at room temperature. 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.
Alpha signal was only detected with AKT3 recombinant protein, while no cross-reactivity was observed with AKT1 and AKT2 proteins. These results demonstrate the specificity of the AKT3 Phospho assay as these three proteins share approximately 80% sequence identity.
