Revvity, 64RBTPEG, HTRF Human Total Rb Detection Kit, 500 Assay Points

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.
HTRF assays offer many advantages over other technologies:

Homogeneous add-and-read format
No wash steps
Low background
Straightforward miniaturization from 96- or 384-well microplates to high density assay formats such as 384-well low volume and 1536-well plates
Stable signal, providing flexibility in time of readout or size of assays
How it works
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. 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-Total
Product Group-Kit
Sample Volume-16 µL
Shipping Conditions-Shipped in Dry Ice
Target Class-Phosphoproteins
Target Species-Human
Technology-TR-FRET
Therapeutic Area-Oncology & Inflammation
Unit Size-500 assay points