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

In HCT-116 cells, Palbociclib efficiently inhibits Retinoblastoma phosphorylation and slightly decreases the total amount of the protein HCT-116 cells were plated at 50 µL in 96-well plates (50,000 cells/well) in complete culture medium, and incubated at 37°C, 5% CO2. After 6 hours, cells were treated with increasing concentrations of Palbociclib (50 µL additional volume) for 19 hours.

After medium removal, cells were then lysed with 50 µL of supplemented lysis buffer for 30 minutes at RT under gentle shaking, and 16 µL of lysate were transferred into a low volume white microplate before the addition of 4 µL of the premixed HTRF Phospho-Rb (Ser780), Phospho-Rb (er807/811), or Total-Rb detection reagents. The HTRF signal was recorded after 4h of 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 780 and Serine 807/811, associated with a decrease in the total amount of the protein (as previously reported by Liu et al, Plos 2017).

HTRF assay compared to Western Blot using Total-Rb cellular assay on human HCT116 cells

Human HCT116 cells were cultured in a T175 flask at 5% CO2, 37°C. At 80% of confluency, cells were lysed and soluble supernatants were collected via centrifugation. Serial dilutions of the cell lysate were performed and 16 µL of each dilution were transferred into a 384-well low volume white microplate, before finally adding Total-Rb cellular kit reagents. A side by side comparison showed the HTRF total assay is at least 27-fold more sensitive than the Western Blot.

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 the 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.