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AlphaLISA SureFire Biotin-Free Human and Mouse Phospho-Rb (Ser807/811) Detection Kit, 500 Assay Points

The AlphaLISA™ SureFire® Biotin-Free Human and Mouse Phospho-Rb (Ser807/811) assay is a sandwich immunoassay for quantitative detection of phospho-Rb (Ser807/811) in cellular lysates using Alpha Technology.

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Feature Specification
Application Cell Signaling
Protocol Time 2h at RT

The AlphaLISA™ SureFire® Biotin-Free Human and Mouse Phospho-Rb (Ser807/811) assay is a sandwich immunoassay for quantitative detection of phospho-Rb (Ser807/811) in cellular lysates using Alpha Technology.

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Save 10% on your first Revvity.com order. Use promo code below.
HELLO10
Product Variants
Unit Size: 100 assay points
Part #:
ASBF-PRB-B-HV
List Price
USD 729.00
Unit Size: 500 assay points
Part #:
ASBF-PRB-B500
List Price
USD 2,463.00
Unit Size: 10,000 assay points
Part #:
ASBF-PRB-B10K
List Price
USD 14,984.00
Unit Size: 50,000 assay points
Part #:
ASBF-PRB-B50K
List Price
USD 48,363.00
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).
USD 2,463.00
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Overview

The retinoblastoma protein (Rb), encoded by RB1, is a tumor suppressor that regulates the cell cycle by inhibiting E2F transcription factors, thereby blocking S phase entry. Rb is inactivated via phosphorylation by cyclin-dependent kinases, enabling cell cycle progression. Mutations in RB1 cause retinoblastoma and are implicated in various cancers, including osteosarcoma and lung cancer, due to the loss of cell cycle control.

The AlphaLISA SureFire Biotin-Free Human and Mouse Phospho-Rb (Ser807/811) Detection Kit is a sandwich immunoassay for the quantitative detection of phospho-Rb 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

Specifications

Application
Cell Signaling
Automation Compatible
Yes
Brand
AlphaLISA SureFire Biotin-Free
Detection Modality
Alpha
Protocol Time
2h at RT
Shipping Conditions
Shipped in Blue Ice
Target
Rb
Target Class
Phosphoproteins
Target Species
Human
Mouse
Technology
Alpha
Therapeutic Area
Oncology
Unit Size
500 assay points

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.

assay principle Phospho AlphaLISA Surefire Ultra Biotin Free

 

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.

2 plates assay protocol AlphaLISA Surefire Ultra Biotin Free Phospho assay.jpg
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.

Phospho-AlphaLISA SureFire Ultra Biotin Free one-plate assay protocol

Assay validation

Decrease of Rb Phospho (Ser807/811) in Palbociclib treated cells

THP-1 cells were seeded in a 96-well plate (200,000 cells/well) in complete RPMI 1640 medium and treated with increasing concentrations of Palbociclib for 24 hours at 37°C, 5% CO2.

After treatment, the cells were lysed with the addition of 50 µL 5X Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Rb Phospho (Ser807/811) and Rb Total levels were evaluated using respective AlphaLISA SureFire Biotin Free assays. For the detection step, lysate was further diluted 1:2 with Lysis Buffer, 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, Palbociclib triggered a dose-dependent decrease in the levels of Phospho (Ser807/811) and Total Rb.

THP 1 cells treated with Palbociclib
Decrease of Rb Phospho (Ser807/811) in CDK6 PROTAC treated cells

THP-1 cells were seeded in a 96-well plate (200,000 cells/well) in complete RPMI 1640 medium and treated with increasing concentrations of BSJ-03-123 (CDK6 PROTAC) for 24 hours at 37°C, 5% CO2.

After treatment, the cells were lysed with the addition of 50 µL 5X Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Rb Phospho (Ser807/811) and ERK1/2 Total levels were evaluated using respective AlphaLISA SureFire Biotin Free assays. For the detection step, lysate was further diluted 1:2 with Lysis Buffer, 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, BSJ-03-123, induced a significant decrease in the levels of Rb Phospho (Ser807/811) while Total ERK1/2 levels remain unchanged.

THP 1 cells treated with CDK6 PROTAC

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