Revvity, 64CDC6S54PEH, HTRF Human and Mouse Phospho-CDC6 (Ser54) Detection Kit, 10,000 Assay Points

Overview
CDC6, or Cell Division Cycle 6, is a member of the AAA+ ATPase family and plays a crucial role in the regulation of DNA replication. It is an essential component of the pre-replication complex, facilitating its assembly at origins of replication to ensure that DNA replication occurs only once per cell cycle. Beyond its role in replication initiation, CDC6 also contributes to the activation and maintenance of cell cycle checkpoints, which are vital for preserving genomic stability.
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
Phospho-CDC6 (Ser54) assay principle
The Phospho-CDC6 (Ser54) assay measures CDC6 phosphorylated at Ser54. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer.
The assay uses two antibodies: one labeled with a donor fluorophore, and the other with an acceptor fluorophore. The first antibody was selected for its specific binding to the phosphorylated motif on the protein, while the second recognizes the protein independently of its phosphorylation state.
Phosphorylation of the protein enables the formation of an immune complex involving both labeled antibodies, bringing the donor fluorophore into close proximity to the acceptor. This interaction generates a FRET signal, whose intensity is directly proportional to the concentration of phosphorylated protein in the sample. The assay provides a reliable means of assessing the protein's phosphorylation state in a no-wash format.

Phospho-CDC6 (Ser54) two-plate assay protocol
The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a 384-well low volume detection plate before the addition of Phospho-CDC6 (Ser54) HTRF detection reagents. This protocol allows the cells' viability and confluence to be monitored.

Phospho-CDC6 (Ser54) one-plate assay protocol
Detection of Phosphorylated CDC6 (Ser54) 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 allows miniaturization while maintaining HTRF quality. Application-Cell Signaling
Brand-HTRF
Detection Modality-HTRF
Lysis Buffer Compatibility-Lysis Buffer 1
Molecular Modification-Phosphorylation
Product Group-Kit
Sample Volume-16 µL
Shipping Conditions-Shipped in Dry Ice
Target-CDC6
Target Class-Phosphoproteins
Target Species-Human Mouse
Technology-TR-FRET
Therapeutic Area-Oncology
Unit Size-10,000 assay points