Revvity, 64CHK2T8PEH, HTRF Human Phospho-CHK-2 (Thr68) Detection Kit, 10,000 Assay Points

Overview
This HTRF cell-based assay enables the rapid, quantitative detection of CHK2 phosphorylated at threonine 68, as a readout of the ATM/CHK2 signaling pathway upon a DNA damage response (DDR)
In response to DNA damage, such as Double Strand Breaks (DSBs), ATM-Chk2 pathway and replication checkpoint responses are activated that mediate G1/S checkpoints to arrest cell cycle progression and allow extra time for DNA repair.
The activation of ATM results in the phosphorylation of the checkpoint kinase Chk2 at threonine 68. Chk2 is a stable protein expressed throughout the cell cycle, and it appears to be largely inactive in the absence of DNA damage. CHK2 activation involves its dimerization and autophosphorylation, and induces the activation of the downstream signal effectors such as the tumor suppressor protein p53 and CdC25C, and also controls cdk2/CyclinA activity.
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
HTRF Phospho CHK2 Thr68 assay principle
The Phospho-CHK2 (Thr68) assay measures CHK2 when phosphorylated at Thr68. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer.
The Phospho-CHK2 (Thr68) assay uses 2 labeled antibodies: one with a donor fluorophore, the other with an acceptor. The first antibody was selected for its specific binding to the phosphorylated motif on the protein, and the second for its ability to recognize the protein independently of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving the two labeled antibodies and which brings the donor fluorophore into close proximity to the acceptor, thereby generating 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 Thr68-CHK2 2-plate assay protocol
The 2 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 additon of the Phospho-CHK2 (Thr68) HTRF detection reagents.
This protocol enables the cells' viability and confluence to be monitored.

 

Phospho-Thr68-CHK2 1-plate assay protocol
Detection of Phosphorylated CHK2 (Thr68) 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
Automation Compatible-Yes
Brand-HTRF
Detection Modality-HTRF
Lysis Buffer Compatibility-Lysis Buffer 1 Lysis Buffer 4
Molecular Modification-Phosphorylation
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-10,000 assay points