-->

HTRF Human Phospho-CHK2 Thr68 Detection Kit HTRF®

This HTRF kit enables the cell-based quantitative detection of CHK2 phosphorylation at Thr68, which is activated upon DNA damage. This kit enables optimal investigation of the ATM/CHK2 pathway, such as via its selective inhibitors.

Voir plus d’informations
  • All inclusive kit All inclusive kit
  • Low sample consumption Low sample consumption
  • No-wash No-wash
  • High sensitivity High sensitivity

This HTRF kit enables the cell-based quantitative detection of CHK2 phosphorylation at Thr68, which is activated upon DNA damage. This kit enables optimal investigation of the ATM/CHK2 pathway, such as via its selective inhibitors.

-

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.

Benefits

  • SPECIFICITY
  • PRECISION

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.

Principle of the HTRF Phospho CHK2 assay

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.

Two-plate protocol of the HTRF phospho CHK2 assay principle

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.

One-plate protocol of the HTRF phospho CHK2 assay principle

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.

Neocarzinostatin dose-response on HEK293 cells

Effect of compounds inducing DNA damage on CHK2 phosphorylation and total protein

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, Hydroxyurea, Doxorubicin, and Etoposid for 2h at 37°C, 5% CO2. The medium was then  removed, and the cells were 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.


The different compounds showed different responses. Neocarzinostatin, Doxorubicin, and Etoposide are known to induce double strand breaks (DSB) and led to phosphorylation of CHK2. On the other hand, Hydroxyurea, which preferably induces single strand breaks (SSB) displayed a partial CHK2 phosphorylation with weak potency. The EC50 of Neocarzinostatin, Doxorubicin, Etoposide and Hydroxyurea were evaluated at 0.1 µM, 1.5 µM, 0.4 µM, and 0.6 mM respectively.


Moreover, the EC80 of Neocarzinostatin was evaluated at 0.3 µM and this concentration was used to assess inhibitors of ATM/CHK2 pathway.

None of the 4 compounds affected the expression level of the CHK2 total protein.

Dose response of DNA damage compounds on CHK2 phosphorylation and total protein
Dose response of DNA damage compounds on CHK2 phosphorylation and total protein

Effect of ATR/CHK1 or ATM/CHK2 pathway inhibitors on HTRF Phospho Thr 68 and total CHK2 kits

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 3 inhibitors of ATR or ATM pathway for 2h at 37 °C, 5% CO2. The cells were then treated with 0.3 µM of Neocarzinostatin (EC80) for another 2h at 37 °C, 5% CO2. The medium was removed, and the 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.


Caffeine is known as a mild ATR/ATM pathway inhibitor, UCN-1 as a potent CHK1 inhibitor, and KU55933 as a selective ATM pathway inhibitor. As expected, UCN-1 had no effect on CHK2 phosphorylation. Caffeine showed a decrease in CHK2 phosphorylation with a weak potency. KU55933 allowed a full inhibition of CHK2 phosphorylation with a higher potency (IC50: 1 µM).


These 3 tested compounds did not affect the expression level of the CHK2 total protein.

Dose response curve of ATR/CHK1 or ATM/CHK2 pathways Inhibitors with HTRF Phospho Thr 68 and total CHK2 kits
Dose response curve of ATR/CHK1 or ATM/CHK2 pathways Inhibitors with HTRF Phospho Thr 68 and total CHK2 kits

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.

Comparison of HTRF phospho Thr68 CHK2 kit with 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.

​​​​​​​
CHK2 signaling pathway

HTRF cellular phospho-protein assays

physiologically relevant results fo fast flowing research - Flyers

Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences

Insider Tips for successful sample treatment - Notes techniques

Optimize your HTRF cell signaling assays on tissues

HTRF and WB compatible guidelines - Notes techniques

Best practices for analyzing tumor xenografts with HTRF phospho assays

Protocol for tumor xenograft analysis with HTRF - Notes techniques

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

HTRF® cell signaling platform combined with iCell® Hepatocytes

A solution for phospho-protein analysis in metabolic disorders - Posters

HTRF phospho-assays reveal subtle drug-induced effects

Detailed protocol and direct comparison with WB - Posters

Universal HTRF® phospho-protein platform: from 2D, 3D, primary cells to patient derived tumor cells

Analysis of a large panel of diverse biological samples and cellular models - Posters

HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts

Tumor xenograft analysis: HTRF versus Western blot - Notes d'application

HTRF cell-based phospho-protein data normalization

Valuable guidelines for efficiently analyzing and interpreting results - Notes d'application

HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers

Increased flexibility of phospho-assays - Notes d'application

HTRF Alpha-tubulin Housekeeping kit

Properly interpret your compound effect - Notes d'application

Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

Analyse of PI3K/AKT/mTor translational control pathway - Notes d'application

How to run a cell based phospho HTRF assay

What to expect at the bench - Vidéos

Unleash the potential of your phosphorylation research with HTRF

A fun video introducing you to phosphorylation assays with HTRF - Vidéos

How to run a cell based phospho HTRF assay

3' video to set up your Phospho assay - Vidéos

Guidelines for Cell Culture and Lysis in Different Formats Prior to HTRF Detection

Seeding and lysing recommendations for a number of cell culture vessels. - Notes techniques

Methodological Aspects of Homogeneous Time-Resolved Fluorescence (HTRF)

Learn how to reduce time and sample consumption - Notes d'application

Assessment of drug efficacy and toxicity by combining innovative technologies

Combination of AlphaLISA®, HTRF®, or AlphaLISA® SureFire® Ultra™ immunoassays with the ATPlite™ 1step cell viability assay - Notes d'application

Plate Reader Requirement

Choosing the right microplate reader ensures you’ll get an optimal readout. Discover our high performance reader, or verify if your lab equipment is going to be compatible with this detection technology.

Let's find your reader