Phospho-Rb (Ser780) assay principle

The Phospho-Rb (Ser780) 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 and 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 (Ser780) two-plate assay protocol

The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates to a low volume detection plate (either HTRF 384-lv or 96-lv plate) before the addition of HTRF Phospho-Rb (Ser780) detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Phospho-Rb (Ser780) one-plate assay protocol

Detection of Phosphorylated Rb (Ser780) 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 Ser780 residue

HCT-116 cells were plated at 50 µL in a 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) 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, associated with a decrease in the total amount of the protein (as previously reported by Liu et al, Plos 2017).

Phospho-Rb cellular assay validation on human and mouse cell lines

NIH 3T3, C2C12, and HTC116 cells were plated at 100,000 cells per well in 96-well plates. After an ON incubation at 37°C, 5% CO2, cell culture medium was removed and 50µL of lysis buffer were added to the cells. A lysis step was carried out, shaking gently for 30 minutes. 16µL of samples were transferred into a 384-well small volume plate, then 4µL of each of the three HTRF Rb detection reagents were added. Signals were recorded after 4 hours.

Both HTRF phospho assays are compatible with mouse models.

HTRF assay compared to Western Blot using Phospho-Rb Ser780 cellular assay on human HCT116 cells

Human HCT116 cells were cultured in T175 flasks 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 Phospho-Ser780 Rb cellular kit reagents. A side by side comparison showed the HTRF Phospho assay is at least 9-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 then 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.