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.

Neocarzinostatin effect on total and phospho Thr 68 CHK2 assay

Human HEK293 cells were plated in a 96-well culture-treated plate (100,000 cells/well) in complete culture medium, and incubated overnight at 37°C, 5% CO2. The cells were treated with a dose-response of Neocarzinostatin for 2h at 37 °C, 5% CO2. Cells were then lysed with 50 µl of supplemented lysis buffer #1 (1X) for 30 min at RT under gentle shaking. After cell lysis, 16 µL of lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF Phospho CHK2 (Thr 68) or Total-CHK2 detection reagents were added. The HTRF signal was recorded after an overnight incubation at room temperature.

As expected, Neocarzinostatin induced single and double strand DNA damage, leading to a dose-dependent increase in CHK2 phosphorylation, without any effect on the expression level of the CHK2 total protein.

HTRF phospho Thr68 CHK2 Assay compared to Western Blot

HEK293 cells were cultured in a T175 flask in complete medium at 37°C, 5% CO2 to confluency.

After medium removal, the cells were lysed with 3 mL of supplemented lysis buffer #1 (1x) for 30 min at RT under gentle shaking.

Serial dilutions of the cell lysate were performed using supplemented lysis buffer #1 (1x), and then 16µL of pure sample and each dilution were transferred into a 384-well small volume microplate before the addition of 4µL of HTRF Phospho Thr68 CHK2 detection reagents. Signals were recorded overnight.

Equal amounts of lysates were loaded into a gel for a side by side comparison between HTRF and Western Blot.

In these conditions, the HTRF phospho Thr68 CHK2 assay is as sensitive as the Western Blot.

ATM/CHK2 and ATR/CHK1 signaling pathways

Double-strand breaks (DSBs) or single-strand breaks (SSBs) are among the most deleterious lesions that threaten genome integrity. These damages can be induced by the effect of cellular metabolites or by DNA-damaging agents such as genotoxics compounds, chemotherapeutic agents, ultraviolet (UV) irradiation or ionizing radiation.

DNA damage response (DDR) is mainly controlled by ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR), two members of the phosphoinositide 3-kinase (PI3K)-related kinase (PIKK) protein kinase family.

In reponse to DNA damage (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.

In response to DNA DSBs, the activation of ATM results in the phosphorylation of the checkpoint kinase Chk2. Chk2 is a stable protein expressed throughout the cell cycle, it appears to be largely inactive in the absence of DNA damage. Its activation involves dimerization and autophosphorylation (Thr383 and Thr 387) and induces the activation of the downstream signal effectors such as the tumor suppressor protein p53, CdC25C and control cdk2/CyclinA activity.

Chemical inhibition of Chk2 during radiation might protect sensitive tissues such as lymphocytes or intestinal epithelium from the side effects of radiotherapy or drugs that cause DSBs. The critical issue here would be to identify suitable inhibitors of Chk2 and test whether this strategy could be applied without increasing the incidence of tumors.

Identification of new small molecule inhibitors of Chk2 and design and validation of novel strategies of checkpoint modulation, combined with the traditional radiation and chemotherapy modalities, hold promise for improved treatment of cancer.