The total Rb kit detects cellular Rb, and can be combined with our phospho-Rb kit for an optimal readout of the pRb-E2F pathway.

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  • No-wash
  • High sensitivity
  • All inclusive kit
  • Low sample consumption

The total Rb kit detects cellular Rb, and can be combined with our phospho-Rb kit for an optimal readout of the pRb-E2F pathway.

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Overview

The Total Rb cell-based assay is designed to monitor the expression level of Rb proteins, both phosphorylated and unphosphorylated. It uses the same buffers as our Phospho-Rb kit, and enables the analysis of phosphorylated and total proteins from a single sample for a better readout of the Cyclin-Cdk pathway. With its central role in cell cycle progression, Rb is an important target for oncological disease studies.

Benefits

  • SPECIFICITY
  • PRECISION
  • DATA NORMALIZATION

Total-Rb assay principle

The Total-Rb assay quantifies the expression level of Rb in a cell lysate. Unlike Western Blot, the assay is entirely plate-based, and does not require gels, electrophoresis, or transfer. The Total-Rb assay uses two labeled antibodies, one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of Rb in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor, and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein’s expression under a no-wash assay format.

Total-Rb 2-plate assay protocol

The 2 plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates to a 384-well low volume detection plate before the addition of Total-Rb HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Total-Rb 1-plate assay protocol

Detection of total Rb 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, Palbociclib efficiently inhibits Retinoblastoma phosphorylation and slightly decreases the total amount of the protein

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 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 total kit / 63ADK106PEG-63ADK106PEH

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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

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Detailed protocol and direct comparison with WB - Posters

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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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