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AlphaLISA SureFire Ultra Human and Mouse Phospho-PRAS40 (Thr246) Detection Kit, 500 Assay Points

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).

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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).

Click to copy promo code to clipboard.
SAVE 15% NOW on online orders. Use promo code below.
YEAREND15
Offer valid until 12/15. Terms and conditions apply.
Product Variants
Unit Size: 100 Assay Points
Part #:
ALSU-PPRAS-A-HV
List Price
USD 694.00
Unit Size: 500 Assay Points
Part #:
ALSU-PPRAS-A500
List Price
USD 2,346.00
Unit Size: 10,000 Assay Points
Part #:
ALSU-PPRAS-A10K
List Price
USD 14,270.00
Unit Size: 50,000 Assay Points
Part #:
ALSU-PPRAS-A50K
List Price
USD 46,060.00

Overview

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:

  • 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

 

Alpha SureFire Ultra kits can be used for:

  • Cellular kinase assays
  • Receptor activation studies
  • Screening

 

Specifications

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

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.

2plates-assay-protocol-AlphaLISA-Surefire-Ultra.jpg
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

Assay validation

Activation of Phospho PRAS40 (Thr246) in Insulin treated cells

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.

Pharmacological Validation (activator) of Phospho (Thr246) PRAS40
Decrease of Phospho PRAS40 (Thr246) in UCN-01 treated cells

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.

Pharmacological Validation (inhibitor) of Phospho (Thr246) PRAS40
Decrease of Phospho PRAS40 (Thr246) in Staurosporine treated cells

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.

Pharmacological Validation (inhibitor) of Phospho (Thr246) PRAS40
Decrease of Phospho PRAS40 (Thr246) in Wortmannin treated cells

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.

Pharmacological Validation (inhibitor) of Phospho (Thr246) PRAS40

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