This HTRF kit enables the cell-based quantitative detection of phosphorylated Rb as a readout of the G0/G1 cell phase transition.
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  • No-wash
  • High sensitivity
  • Ready-to-use
  • Low sample consumption
This HTRF kit enables the cell-based quantitative detection of phosphorylated Rb as a readout of the G0/G1 cell phase transition.
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Overview

This HTRF cell-based assay conveniently and accurately quantifies phosphorylated Retinoblastoma protein at Ser807/811. Rb belongs to the pocket protein family, and acts as a tumor suppressor regulating cell cycle progression. The active underphosphorylated Rb form interacts and represses transcriptional activity of E2F factors, thereby preventing G0/G1 cell cycle transition and progression in the cell cycle.  In the presence of mitogenic signals, cyclin D-CDK4/6, followed by cyclin E-CDK2, sequentially phosphorylate and inactivate Rb, releasing the E2F transcription factors required for cell cycle progression.

Benefits

  • SPECIFICITY
  • PRECISION

Phospho-Rb (Ser807/811) assay principle

The Phospho-Rb (Ser807/811) assay measures Rb when phosphorylated at Ser780. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Phospho-Rb (Ser780) assay uses 2 labeled antibodies, one with a donor fluorophore, the other with an acceptor. The first antibody is selected for its specific binding to the phosphorylated motif on the protein, the second for its ability to recognize the protein independent of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving the two labeled antibodies, which brings the donor fluorophore into close proximity to the acceptor, and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of phosphorylated protein present in the sample, and provides a means of assessing the protein's phosphorylation state under a no-wash assay format.

Phospho-Rb (Ser807/811) two-plate assay protocol

The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a low volume detection plate (either HTRF 384-lv or 96-lv plate) before the addition of HTRF Phospho-Rb (Ser807/811) detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Phospho-Rb (Ser807/811) one-plate assay protocol

Detection of Phosphorylated Rb (Ser807/811) with HTRF reagents can be performed in a single plate used for culturing, stimulation, and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.

In HCT-116 cells, the CDK4/CDK6 inhibitor, Palbociclib, efficiently inhibits Retinoblastoma phosphorylation on Ser807/811 residue

HCT-116 cells were plated under 100 µL in a 96-well plates (40,000 cells/well) in complete culture medium and incubated at 37°C, 5% CO2. The day after, the medium was removed and cells were treated with increasing concentrations of Palbociclib (100 µL for 24 hours).

After medium removal, cells were then lysed with 50 µL of supplemented lysis buffer #1 for 30 minutes at RT under gentle shaking, and 16 µL of lysate were transferred twice over into a low volume white microplate before the addition of 4 µL of the premixed HTRF phospho-Rb (Ser807/811) or Total-Rb detection reagents. The HTRF signal was recorded after ON incubation.


Treatment with Palbociclib, a Cyclin-Dependent Kinase (Cdk4 and Cdk 6) inhibitor, leads to a significant decrease in the phosphorylation of Rb on Serine 807/811, associated with a slight decrease in the total amount of the protein.

(as previously reported by Liu et al, Plos 2017)

The Retinoblastoma protein in the cell-division cycle

The 110 kDa Retinoblastoma protein Rb belongs to the pocket protein family comprising p107 and p130.

Rb acts as a tumor suppressor regulating cell cycle progression.

Mutations inactivating the protein result in the development of retinoblastoma cancer, where retinal cells are not replaced and are subjected to high levels of mutagenic UV radiation.

In the absence of mitogenic signals, active underphosphorylated Rb binds and inhibits the E2F transcription factors which are required for the entry into S phase.

By keeping E2F inactivated, Rb maintains the cell in the G1 phase, preventing progression through the cell cycle.

In the presence of mitogenic signals, Rb is sequentially phosphorylated and inactivated by cyclin D-CDK4/6, and cyclin E-CDK2. This phosphorylation event induces the release of E2F transcription factors.

Finally, E2F activates the transcription of genes such as cyclins, Cdk, Thymidine kinase, or PCNA, which play essential roles in DNA synthesis and replication, as well as in cell division.

Cisbio Product Catalog 2019

All your HTRF assays in one document! - Catalog

A guide to Homogeneous Time Resolved Fluorescence

General principles of HTRF - Guides

Product Insert RB-P-S807/811 kit / 63ADK105PEG-63ADK105PEH

63ADK105PEG-63ADK105PEH - Product Insert

How HTRF compares to Western Blot and ELISA

Get the brochure about technology comparison. - Brochures

Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences

Insider Tips for successful sample treatment - Technical Notes

Optimize your HTRF cell signaling assays on tissues

HTRF and WB compatible guidelines - Technical Notes

Best practices for analyzing tumor xenografts with HTRF phospho assays

Protocol for tumor xenograft analysis with HTRF - Technical Notes

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

HTRF® cell signaling platform combined with iCell® Hepatocytes

A solution for phospho-protein analysis in metabolic disorders - Posters

HTRF phospho-assays reveal subtle drug-induced effects

Detailed protocol and direct comparison with WB - Posters

Universal HTRF® phospho-protein platform: from 2D, 3D, primary cells to patient derived tumor cells

Analysis of a large panel of diverse biological samples and cellular models - Posters

HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts

Tumor xenograft analysis: HTRF versus Western blot - Application Notes

HTRF cell-based phospho-protein data normalization

Valuable guidelines for efficiently analyzing and interpreting results - Application Notes

HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers

Increased flexibility of phospho-assays - Application Notes

HTRF Alpha-tubulin Housekeeping kit

Properly interpret your compound effect - Application Notes

Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

Analyse of PI3K/AKT/mTor translational control pathway - Application Notes

Side-by-side comparison of HTRF, Western Blot, ELISA and AlphaScreen® SureFire®

Do all cell-based kinase assays perform similarly? - Posters

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