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HTRF Human/Mouse Phospho-CHK1 Ser345 Detection Kit HTRF®

This HTRF kit enables the cell-based quantitative detection of CHK1 phosphorylation at Ser345, which is activated upon DNA damage. This kit enables optimal investigation of the ATR/CHK1 pathway, such as via its selective inhibitors.

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  • 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 CHK1 phosphorylation at Ser345, which is activated upon DNA damage. This kit enables optimal investigation of the ATR/CHK1 pathway, such as via its selective inhibitors.

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Overview

This HTRF cell-based assay enables the rapid, quantitative detection of CHK1 phosphorylated at Serine 345, as a readout of the ATR/CHK1 signaling pathway upon a DNA damage response (DDR)

In response to DNA damage, for example Single Strand Breaks (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 of ssDNA-RPA through its interacting partner, ATRIP, which leads to Chk1 phosphorylation and activation and in turn induces downstream signaling.


Benefits

  • SPECIFICITY
  • PRECISION

HTRF phospho CHK1 Ser 345 assay principle

The Phospho-CHK1 (Ser345) assay measures CHK1 when phosphorylated at Ser345. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer.

The Phospho-CHK1 (Ser345) 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 CHK1 Ser345 assay

Phospho CHK1 Ser 345 2-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 additon of the Phospho-CHK1 (Ser345) HTRF detection reagents.

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

 Description de la ressource:

Phospho CHK1 Ser 345 1-plate assay protocol

Detection of Phosphorylated CHK1 (Ser345) 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 CHK1 Ser 345 assay principle

Human/Mouse CHK1 assay compatibility: Neocarzinostatin dose response curves

Human HEK293 cells or Mouse NIH/3T3 cells were plated in 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 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 CHK1 (Ser 345) or Total-CHK1 detection reagents were added. The HTRF signal was recorded after an overnight incubation at room temperature.


As expected, Neocarzinostatin induced single strand DNA damage, leading to a dose-dependent increase in CHK1 phosphorylation, without any effect on the expression level of the total protein in either the human or the mouse cell lines.

Neocarzinostatin dose-response on HEK293 cells or NIH/3T3
Neocarzinostatin dose-response on HEK293 cells or NIH/3T3

Effect of compounds inducing DNA damage on CHK1 phosphorylation and on 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 CHK1 (Ser 345) or Total-CHK1 detection reagents were added. The HTRF signal was recorded after an overnight incubation at room temperature.


The different compounds showed different responses. Neocarzinostatin, hydroxyurea, and etoposide, which induce single strand breaks (SSB), resulted in the full phosphorylation of CHK1. Doxorubicin, that favors double strand breaks (DSB), displayed a partial CHK1 phosphorylation. The EC50 of Neocarzinostatin, hydroxyurea, etoposide, and Doxorubicin were assessed at 1.2 µM, 0.1 mM, 3.3 µM, and 7.1 µM respectively.

Moreover, the EC80 of Neocarzinostatin was measured at 3 µM, and this concentration was used to assess inhibitors of the ATR/CHK1 Pathway.

On the other hand, these 4 compounds did not affect the expression level of the CHK1 total protein.

Dose response of DNA damage compounds on CHK1 phosphorylation and total protein
Dose response of DNA damage compounds on CHK1 phosphorylation and total protein

Effect of ATR/CHK1 or ATM/CHK2 pathway inhibitors on CHK1 phosphorylation and on 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 3 inhibitors of the ATR or ATM pathways for 2h at 37 °C, 5% CO2. The cells were then treated with 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 CHK1 (Ser 345) or Total-CHK1 detection reagents were added. The HTRF signal was recorded after an overnight incubation at room temperature.


Caffeine is known to be a mild ATR/ATM pathway inhibitor, UCN-1 is a potent CHK1 inhibitor, and KU55933 is an ATM pathway inhibitor only. As expected, UCN-1 allowed the full inhibition of the CHK1 phosphorylation with high potency (IC50 15 nM), and the caffeine displayed a weaker potency. The KU55933 compound did not induce CHK1 phosphorylation inhibition.


On the other hand, none of these 3 tested compounds affected the expression level of the CHK1 total protein.

Dose response curve of ATR/CHK1 or ATM/CHK2 pathways Inhibitors with HTRF Phospho Ser 365 and total CHK1 kits
Dose response curve of ATR/CHK1 or ATM/CHK2 pathways Inhibitors with HTRF Phospho Ser 365 and total CHK1 kits

HTRF phospho CHK1 (ser345) 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 of each dilution were transferred into a 384-well small volume microplate, before the addition of 4µL of HTRF Phospho Ser345 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 Phospho Ser345 CHK1 assay is as sensitive as the Western Blot.

Comparison of HTRF Phospho CHK1 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 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 tract 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, the cells will not elicit cell cycle arrest for DNA repair, thus leading to cancer cell death.  Inhibiting the ATR pathway has therefore 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.

CHK1 signaling pathway

HTRF cellular phospho-protein assays

Physiologically relevant results fo fast flowing research - Flyers

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Insider Tips for successful sample treatment - Technical Notes

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HTRF and WB compatible guidelines - Technical Notes

Best practices for analyzing tumor xenografts with HTRF phospho assays

Protocol for tumor xenograft analysis with HTRF - Technical Notes

Key guidelines to successful cell signaling experiments

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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 - Application Notes

HTRF cell-based phospho-protein data normalization

Valuable guidelines for efficiently analyzing and interpreting results - Application Notes

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

Increased flexibility of phospho-assays - Application Notes

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How to run a cell based phospho HTRF assay

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Unleash the potential of your phosphorylation research with HTRF

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How to run a cell based phospho HTRF assay

3' video to set up your Phospho assay - Videos

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

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Learn how to reduce time and sample consumption - Application Notes

Assessment of drug efficacy and toxicity by combining innovative technologies

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Product Insert CHK1 p-S345 Kit / 64CHK1S5PEG-64CHK1S5PEH

64CHK1S5PEG-64CHK1S5PEH - Product Insert

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