Phospho-c-RAF (Ser338) cellular kit
Convenient, fast assay quantifying Phoshpo-c-RAF (Ser338) modulation
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This HTRF cell-based assay conveniently and accurately quantifies phosphorylated Retinoblastoma protein at Ser780. 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 the 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.
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.
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.
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.
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).
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 recored after 4 hours.
Both HTRF phospho assays are compatible with mouse models.
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 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.
HTRF Product Catalog 2020 July update
All your HTRF assays in one document! - Catalog
A guide to Homogeneous Time Resolved Fluorescence
General principles of HTRF - Guides
Key guidelines to successful cell signaling experiments
Mastering the art of cell signaling assays optimization - Guides
Optimize your HTRF cell signaling assays on tissues
HTRF and WB compatible guidelines - Technical Notes
HTRF Alpha-tubulin Housekeeping kit
Properly interpret your compound effect - Application Notes
Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences
Insider Tips for successful sample treatment - Technical Notes
HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers
Increased flexibility of phospho-assays - Application Notes
HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts
Tumor xenograft analysis: HTRF versus Western blot - Application Notes
Best practices for analyzing tumor xenografts with HTRF phospho assays
Protocol for tumor xenograft analysis with HTRF - Technical Notes
Unleash the potential of your phosphorylation research with HTRF
Unmatched ease of use, sensitivity and specificity assays - Videos
How to run a cell based phospho HTRF assay
What to expect at the bench - Videos
Cisbio lysis buffer compatibility
Cell Signaling: Biomarkers, Phospho- & total-protein Assays - Flyers
Cell Signaling: Biomarkers, Phospho- & total-protein assays - Flyers
PI3K/AKT/mTor translational control pathway - Posters
Product Insert RB P-S780 kit / 63ADK104PEG-63ADK104PEH
63ADK104PEG-63ADK104PEH - Product Insert
How HTRF compares to Western Blot and ELISA
Get the brochure about technology comparison. - Brochures
Multi-tissue cellular modeling and anlysis of insulin signaling - Posters
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
A single technology for 2D cells, 3D cells, and xenograft models - 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
From 2D, 3D cell cultures to xenografts: A smart HTRF platform to maximize anticancer drug discovery
One technology across all samples - Application Notes
HTRF cell-based phospho-protein data normalization
Valuable guidelines for efficiently analyzing and interpreting results - Application Notes
Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling
Analyse of PI3K/AKT/mTor translational control pathway - Application Notes
Cell-based kinase assays in HTS ? potential and limitations for primary and secondary screening
In collaboration with Bayer - Scientific Presentations
Unleash the potential of your phosphorylation research with HTRF
A fun video introducing you to phosphorylation assays with HTRF - Videos
How to run a cell based phospho HTRF assay
3' video to set up your Phospho assay - Videos
HTRF cellular phospho-protein assays
Physiologically relevant results fo fast flowing research - Flyers
Product Insert RB P-S780 kit / 64RBS780PEG-64RBS780PEH
64RBS780PEG-64RBS780PEH - Product Insert
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