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AlphaLISA SureFire Ultra Human Phospho-AKT1/2/3 (Ser473) Detection Kit, 100 Assay Points

AlphaLISA SureFire Ultra Phospho-Protein image
AlphaLISA SureFire Ultra Detection Kit
AlphaLISA phospho RET detection kit View All
AlphaLISA SureFire Ultra Human Phospho-AKT1/2/3 (Ser473) Detection Kit, 100 Assay Points
AlphaLISA SureFire Ultra Phospho-Protein image
AlphaLISA SureFire Ultra Detection Kit
AlphaLISA phospho RET detection kit
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Video Play Button
AlphaLISA SureFire Ultra Phospho-Protein image
AlphaLISA SureFire Ultra Detection Kit
AlphaLISA phospho RET detection kit
AlphaLISA SureFire Ultra Phospho-Protein
AlphaLISA SureFire Ultra Phospho-Protein image
AlphaLISA SureFire Ultra Detection Kit
AlphaLISA phospho RET detection kit
AlphaLISA SureFire Ultra Phospho-Protein image
AlphaLISA SureFire Ultra Detection Kit
AlphaLISA phospho RET detection kit
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The AlphaLISA™ SureFire® Ultra™ p-AKT1/2/3 (Ser473) assay is a sandwich immunoassay for quantitative detection of phospho-AKT1/2/3 (phosphorylated on Ser473) in cellular lysates using Alpha Technology.

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Feature Specification
Application Cell Signaling
Sample Volume 30 µL

The AlphaLISA™ SureFire® Ultra™ p-AKT1/2/3 (Ser473) assay is a sandwich immunoassay for quantitative detection of phospho-AKT1/2/3 (phosphorylated on Ser473) in cellular lysates using Alpha Technology.

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Product Variants
Unit Size: 500 assay points
Part #:
ALSU-PAKT-B500
Unit Size: 10,000 assay points
Part #:
ALSU-PAKT-B10K
Unit Size: 50,000 assay points
Part #:
ALSU-PAKT-B50K
Unit Size: 100 assay points
Part #:
ALSU-PAKT-B-HV
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).
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AlphaLISA SureFire Ultra Phospho-Protein image
AlphaLISA SureFire Ultra Detection Kit
AlphaLISA phospho RET detection kit
AlphaLISA SureFire Ultra Phospho-Protein image
AlphaLISA SureFire Ultra Detection Kit
AlphaLISA phospho RET detection kit
Video Play Button
Video Play Button
AlphaLISA SureFire Ultra Human Phospho-AKT1/2/3 (Ser473) Detection Kit, 100 Assay Points
AlphaLISA SureFire Ultra Phospho-Protein image
AlphaLISA SureFire Ultra Detection Kit
AlphaLISA phospho RET detection kit
Video Play Button
Video Play Button
AlphaLISA SureFire Ultra Phospho-Protein image
AlphaLISA SureFire Ultra Detection Kit
AlphaLISA phospho RET detection kit
AlphaLISA SureFire Ultra Phospho-Protein
AlphaLISA SureFire Ultra Human Phospho-AKT1/2/3 (Ser473) Detection Kit, 100 Assay Points
AlphaLISA SureFire Ultra Phospho-Protein image
AlphaLISA SureFire Ultra Detection Kit
AlphaLISA phospho RET detection kit
AlphaLISA SureFire Ultra Phospho-Protein image
AlphaLISA SureFire Ultra Detection Kit
AlphaLISA phospho RET detection kit
Video Play Button
Video Play Button

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OptiPlate 384 image

OptiPlate 384-well, white opaque, case of 50

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OptiPlate 384 image

OptiPlate 384-well, white opaque, case of 200

Part #: 6007299

Product information

Overview

The AKT serine/threonine kinase, also known as protein kinase B (PKB), is a crucial component of the PI3K/AKT/mTOR signaling pathway. In humans, there are three AKT isoforms (AKT1, AKT2, and AKT3) encoded by separate genes. AKT plays a central role in various cellular processes, including cell survival, proliferation, metabolism, and angiogenesis. Activation of AKT occurs through phosphorylation at two key sites: Thr308 and Ser473. The phosphorylation of Ser473 is particularly important for full activation of AKT and its downstream signaling. AKT signaling is frequently dysregulated in various diseases, especially cancer, where hyperactivation of the pathway contributes to tumor growth, metastasis, and therapy resistance.

The AlphaLISA SureFire Ultra Human Phospho-AKT1/2/3 (Ser473) Detection Kit is a sandwich immunoassay designed for the quantitative detection of phosphorylated AKT (Ser473) 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.

In the AlphaLISA™ SureFire® Ultra™ assay, Donor beads are coated with streptavidin to capture one of the antibodies, which is biotinylated. Acceptor beads are coated with a proprietary CaptSure™ agent that immobilizes the other antibody, labeled with a CaptSure™ tag. In the presence of phosphorylated protein, the two antibodies bring the Donor and Acceptor beads close together, generating signal. The amount of light emission is directly proportional to the amount of phosphoprotein present in the sample.

AlphaLISA SureFire Ultra kits are compatible with
  • Cell and tissue lysates
  • Antibody modulators
  • Biotherapeutic antibodies
Alpha SureFire kits can be used for
  • Cellular kinase assays
  • Receptor activation studies
  • Screening

Specifications

Application
Cell Signaling
Automation Compatible
Yes
Brand
AlphaLISA SureFire Ultra
Cellular or Signaling Pathway
PI3K/Akt/mTOR
Detection Modality
Alpha
Lysis Buffer Compatibility
Lysis Buffer
Molecular Modification
Phosphorylation
Product Group
Kit
Sample Volume
30 µL
Shipping Conditions
Shipped in Blue Ice
Target
Akt1/2/3
Target Class
Phosphoproteins
Target Species
Human
Technology
Alpha
Therapeutic Area
Cardiovascular
Central Nervous System
Metabolic
Unit Size
100 assay points

Image gallery

AlphaLISA SureFire Ultra Phospho-Protein image
AlphaLISA SureFire Ultra Detection Kit
AlphaLISA phospho RET detection kit
AlphaLISA SureFire Ultra Phospho-Protein image
AlphaLISA SureFire Ultra Detection Kit
AlphaLISA phospho RET detection kit
AlphaLISA SureFire Ultra Human Phospho-AKT1/2/3 (Ser473) Detection Kit, 100 Assay Points
AlphaLISA SureFire Ultra Phospho-Protein image
AlphaLISA SureFire Ultra Detection Kit
AlphaLISA phospho RET detection kit
AlphaLISA SureFire Ultra Phospho-Protein image
AlphaLISA SureFire Ultra Detection Kit
AlphaLISA phospho RET detection kit
AlphaLISA SureFire Ultra Human Phospho-AKT1/2/3 (Ser473) Detection Kit, 100 Assay Points
AlphaLISA SureFire Ultra Phospho-Protein image
AlphaLISA SureFire Ultra Detection Kit
AlphaLISA phospho RET detection kit
AlphaLISA SureFire Ultra Phospho-Protein image
AlphaLISA SureFire Ultra Detection Kit
AlphaLISA phospho RET detection kit

Video gallery

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AlphaLISA SureFire Ultra Human Phospho-AKT1/2/3 (Ser473) Detection Kit, 100 Assay Points
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AlphaLISA SureFire Ultra Human Phospho-AKT1/2/3 (Ser473) Detection Kit, 100 Assay Points
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Citations

How it works

Phospho-AlphaLISA SureFire Ultra assay principle

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.

Assay Principle Phospho AlphaLISA SureFire Ultra

 

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 permits the cells' viability and confluence to be monitored. In addition, lysates from a single well can be used to measure multiple targets.

2 Plates Assay Protocol AlphaLISA Surefire Ultra Phospho Assay
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 AlphaLISA SureFire Ultra quality.

1 plate assay protocol AlphaLISA Surefire Ultra Phospho assay

Assay validation

Activation of AKT1/2/3 phospho-(Ser473) in Insulin treated cells

HeLa 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 serum starved for 3 hours and 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). AKT1/2/3 Phospho (Ser473) 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 an increase in levels of Phospho (Ser473) AKT1/2/3 while Total levels remained unchanged.

HeLa cells treated with Insulin
Activation of AKT1/2/3 phospho-(Ser473) in Razuprotafib treated cells

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 serum 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). AKT1/2/3 Phospho (Ser473) 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 an increase in the level of Phospho (Ser473) AKT1/2/3 while Total levels remained unchanged.

HUVEC cells treated with Razuprotafib
Inhibition of AKT1/2/3 phospho-(Ser473) in Wortmannin treated cells

HEK293 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 treated 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). AKT1/2/3 Phospho (Ser473) 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, Wortmannin triggered a decrease in the levels of Phospho (Ser473) AKT1/2/3 while Total levels remained unchanged.

HEK293 cells treated with Wortmannin
Inhibition of AKT1/2/3 phospho-(Ser473) in Torin1 treated cells

MCF7 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 Torin1 for 18 hours.

After treatment, the cells were lysed with 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 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, Torin1 triggered a decrease in the levels of Phospho (Ser473) AKT1/2/3 while Total levels remained unchanged.

MCF7 cells treated with Torin1

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