Revvity, 64RBS780PEH, HTRF Human and Mouse Phospho-Rb (Ser780) Detection Kit, 10,000 Assay Points

Overview
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.
How it works
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. Application-Cell Signaling
Brand-HTRF
Detection Modality-HTRF
Lysis Buffer Compatibility-Lysis Buffer 1 Lysis Buffer 2 Lysis Buffer 3 Lysis Buffer 4 Lysis Buffer 5
Molecular Modification-Phosphorylation
Product Group-Kit
Sample Volume-16 µL
Shipping Conditions-Shipped in Dry Ice
Target Class-Phosphoproteins
Target Species-Human Mouse
Technology-TR-FRET
Therapeutic Area-Oncology & Inflammation
Unit Size-10,000 assay points