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.

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.

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.

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.

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.