HTRF Human and Mouse Total CHK-1 Detection Kit, 500 Assay Points

HTRF Total CHK1 assay principle

The Total-CHK1 assay quantifies the expression level of CHK1 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer.

The Total-CHK1 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 CHK1 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.

HTRF Total-CHK1 2-plate assay protocol

The two-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 the Total-CHK1 HTRF detection reagents.

This protocol enables the cells' viability and confluence to be monitored.

Total-CHK1 1-plate assay protocol

Detection of Total CHK1 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.

HTRF Total CHK1 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 16µL of pure sample and each dilution were transferred into a 384-well small volume microplate before the addition of 4µL of HTRF Total CHK1 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 total CHK1 assay is at least 8-fold more sensitive than 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 can threaten genome integrity. These kinds of damage can be induced by the effect of cellular metabolites or by DNA-damaging agents such as genotoxic 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 response to DNA damage (SSBs), the ATR–Chk1 pathway and replication checkpoint responses are activated, mediating G2/M checkpoints to arrest cell cycle progression and allow extra time for DNA repair. ATR is recruited to track ssDNA-RPA through its interacting partner, ATRIP, which leads to Chk1 phosphorylation and activation. CHK1 activation involves the signal transduction pathway up to the modulation of cdc2/cyclin B1 activity and the control of Cdc25A.

Many cancers are deficient in the G1/S checkpoint, including those with defective p53. If the ATR pathway is inhibited in G1 checkpoint-deficient cells due to DNA damage, cells will not elicit cell cycle arrest for DNA repair, leading to cancer cell death. Thus inhibiting the ATR pathway has become an appealing strategy to selectively sensitize p53-deficient cells to chemotherapeutic agents that damage DNA.

The identification of innovative small molecule Chk1 and Chk2 inhibitors and the validation of novel strategies of checkpoint modulation, combined with the traditional radiation and chemotherapy modalities, hold promise for improved treatment of cancer.