-->

HTRF Human Phospho-ATM Ser1981 Detection Kit HTRF®

This HTRF kit enables the cell-based quantitative detection of ATM phosphorylation at Ser1981, which is activated upon DNA damage. This kit enables optimal investigation of the ATM/CHK2 pathway, including selective inhibitors.

See more
  • 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 ATM phosphorylation at Ser1981, which is activated upon DNA damage. This kit enables optimal investigation of the ATM/CHK2 pathway, including selective inhibitors.

-

Overview

This HTRF cell-based assay enables the rapid, quantitative detection of ATM phosphorylated at Serine 1981, as a readout of the ATM/CHK2 signaling pathway upon a DNA damage response (DDR). This kit is complementary to the phospho CHK2 Thr68 for studies of the ATM-CHK2 pathway.

In response to DNA damage, such as Double Strand Breaks (DSBs), the ATM–Chk2 pathway and replication checkpoint responses are activated. These mediate G1/S checkpoints to arrest cell cycle progression and allow extra time for DNA repair.

In response to DNA DSBs, ATM autophosphorylates to generate the active monomeric kinase. Activation of ATM results in the phosphorylation of a diverse array of downstream targets such as H2A histone family member X, H2Ax, or the checkpoint kinase Chk2 and its downstream effectors.

Benefits

  • SPECIFICITY
  • PRECISION

HTRF phospho ATM assay principle

The Phospho-ATM (Ser1981) assay measures ATM when phosphorylated at Serine 1981. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer.

The Phospho-ATM (Ser 1981) 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 ATM assay

HTRF Phospho-ATM Ser 1981 2-plate assay protocol

The 2 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 phospho-ATM  ser 1981 HTRF detection reagents.

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

Two-plate protocol of the HTRF phospho ATM assay principle

HTRF Phospho-ATM Ser 1981 one-plate assay protocol

Detection of phospho ATM Ser 1981 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 ATM assay principle

Neocarzinostatin effect on total and phospho Ser 1981 ATM 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. Cells were treated with a dose-response of neocarzinostatin 2h at 37 °C, 5% CO2. They 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 ATM (Ser 1981) or Total-ATM 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 ATM phosphorylation, without any effect on the expression level of the ATM total protein.

Neocarzinostatin dose-response on HEK293 cells

Effect of compounds inducing DNA damage on ATM phosphorylation (Ser1981) 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. Cells were treated with a dose-response of neocarzinostatin, hydroxyurea, doxorubicin, and etoposide, 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 ATM (Ser1981) or Total-ATM 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 here led to phosphorylation of ATM. On the other hand hydroxyurea, which preferably induces single strand breaks (SSBs), displayed a partial ATM phosphorylation with weak potency. The EC50 of neocarzinostatin, doxorubicin, etoposide, and hydroxyurea were evaluated at 0.2 µM, 1.4 µM, 0.9 µM, and 0.3 mM respectively.

Moreover, the EC80 of neocarzinostatin was evaluated at 1 µM, and this concentration was used to assess inhibitors of the ATM/CHK2 Pathway.

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

Dose response of DNA damage compounds on ATM phosphorylation (Ser1981) and total protein
Dose response of DNA damage compounds on ATM phosphorylation (Ser1981) and total protein

Effect of ATR/CHK1 or ATM/CHK2 pathway inhibitors on HTRF Phospho Ser1981 and Total ATM 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. 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 1 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 ATM Ser 1981 or Total-ATM 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 a selective ATM pathway inhibitor. As expected, UCN-1 had no effect on ATM phosphorylation. Caffeine showed a decrease in ATM phosphorylation with weak potency. KU55933 allowed a full inhibition of ATM phosphorylation with higher potency.

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

Dose response curve of ATR/CHK1 or ATM/CHK2 pathways Inhibitors with HTRF Phospho Ser1981 and total ATM kits
Dose response curve of ATR/CHK1 or ATM/CHK2 pathways Inhibitors with HTRF Phospho Ser1981 and total ATM kits

ATM/CHK2 and ATR/CHK1 signaling pathways

DNA double-strand breaks (DSBs) or single-strand breaks (SSBs) are among the most deleterious lesions that threaten genome integrity. They 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 by 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 (DSBs), ATM–Chk2 pathway and replication checkpoint responses are activated, mediating G1/S checkpoints to arrest cell cycle progression and allow extra time for DNA repair.

Reacting to DNA DSBs, ATM autophosphorylates to generate the active monomeric kinase. Activation of ATM results in the phosphorylation of a diverse array of downstream targets such as H2A histone family member X, H2Ax, or the checkpoint kinase Chk2 and its downstream effectors.

The isolation of new small molecule inhibitors of ATM, and the design and validation of novel strategies of checkpoint modulation, combined with the traditional radiation and chemotherapy modalities, hold promise for improved treatment of cancers.

ATM 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 - Technical Notes

Optimize your HTRF cell signaling assays on tissues

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

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 - 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

HTRF Alpha-tubulin Housekeeping kit

Properly interpret your compound effect - Application Notes

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

Analyse of PI3K/AKT/mTor translational control pathway - Application Notes

How to run a cell based phospho HTRF assay

What to expect at the bench - Videos

Unleash the potential of your phosphorylation research with HTRF

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

How to run a cell based phospho HTRF assay

3' video to set up your Phospho assay - Videos

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

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

Methodological Aspects of Homogeneous Time-Resolved Fluorescence (HTRF)

Learn how to reduce time and sample consumption - Application Notes

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 - Application Notes

Product Insert ATM p-S1981 Kit / 64ATMS1PEG-64ATMS1PEH

64ATMS1PEG-64ATMS1PEH - Product Insert

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