Phospho-ATM (S1981) Recombinant Monoclonal Antibody

What is the biological significance of ATM phosphorylation at S1981?

ATM (Ataxia Telangiectasia Mutated) protein kinase undergoes autophosphorylation at serine 1981 (S1981) as part of its activation mechanism following DNA double-strand breaks. This phosphorylation event is considered a hallmark of ATM activation in the DNA damage response pathway. When cells are exposed to ionizing radiation or radiomimetic agents, ATM dimers rapidly autophosphorylate at S1981, causing the dissociation of the dimers into active monomers that can then phosphorylate downstream substrates involved in cell cycle checkpoints and DNA repair . This phosphorylation is one of several critical modifications that regulate ATM's function as a master regulator of cellular responses to DNA damage .

How does the Phospho-ATM (S1981) antibody specifically recognize the phosphorylated form?

The Phospho-ATM (S1981) antibody is designed to recognize only the phosphorylated epitope surrounding serine 1981 of the ATM protein. This specificity is achieved through careful immunization strategies using synthetic phosphopeptides derived from the human ATM sequence surrounding the S1981 residue . The recombinant monoclonal antibodies are produced by first immunizing animals with these phosphopeptides, followed by isolation of positive splenocytes, RNA extraction, and reverse transcription to obtain the antibody gene sequence . The heavy and light chain sequences are then cloned into expression vectors and transfected into mammalian cells for production. The resulting antibodies undergo affinity purification to ensure high specificity for the phosphorylated form of ATM at S1981, with minimal cross-reactivity to the non-phosphorylated form .

What are the recommended experimental applications for this antibody?

The Phospho-ATM (S1981) Recombinant Monoclonal Antibody has been validated for several experimental applications:

For optimal results in Western blot applications, protocols typically recommend using PVDF membranes and specific buffer conditions. For example, detection has been demonstrated using HeLa cell lysates treated with DNA-damaging agents like camptothecin (CPT) . When conducting immunofluorescence studies, the antibody can effectively visualize the nuclear distribution pattern of phospho-ATM and its colocalization with other DNA damage markers such as γH2AX .

How can the Phospho-ATM (S1981) antibody be used to investigate the temporal dynamics of ATM activation?

To investigate the temporal dynamics of ATM activation, researchers can design time-course experiments using the Phospho-ATM (S1981) antibody. Based on published research, the following methodological approach is recommended:

• Treat cells with DNA-damaging agents such as ionizing radiation (1-10 Gy), radiomimetic drugs (neocarzinostatin, camptothecin), or oxidative stress inducers.

• Collect samples at multiple time points (e.g., 5, 15, 30 minutes, 1, 2, 6, 24 hours post-treatment).

• Process samples for Western blotting or immunofluorescence using the Phospho-ATM (S1981) antibody.

• Quantify the phosphorylation signal relative to total ATM levels using appropriate imaging software.

This approach reveals that ATM phosphorylation at S1981 occurs rapidly (within minutes) after DNA damage and may persist for several hours, with different kinetics depending on the damage type and cell context . The immunofluorescence application is particularly valuable for observing the transition from diffuse nuclear staining to discrete foci that co-localize with DNA damage sites, which typically occurs within 30 minutes to 6 hours post-damage .

What is the relationship between ATM S1981 phosphorylation and other ATM phosphorylation sites?

Recent research has identified multiple functionally important phosphorylation sites in ATM beyond S1981, including S367 and S1893. These sites show distinct yet interdependent patterns of phosphorylation following DNA damage:

Experimental approaches to study these relationships include:

• Comparing phosphorylation kinetics using site-specific antibodies

• Generating phosphorylation site mutants (S367A, S1893A, S1981A)

• Assessing interdependence by examining how mutation at one site affects phosphorylation at others

Research indicates that mutation of any single phosphorylation site (S367A, S1893A, or S1981A) reduces but does not completely eliminate ATM kinase activity, suggesting partially overlapping but non-redundant functions . For comprehensive investigation, researchers should consider examining all phosphorylation sites simultaneously, as focusing solely on S1981 may provide an incomplete picture of ATM activation status.

How does the Mre11-Rad50-Nbs1 complex influence ATM S1981 phosphorylation?

The Mre11-Rad50-Nbs1 (MRN) complex plays a critical role in facilitating ATM S1981 phosphorylation following DNA damage. To experimentally investigate this relationship:

• Deplete MRN components using siRNA targeting Mre11, Rad50, or Nbs1

• Use cell lines with deficiencies in MRN components (e.g., NBS cells lacking functional Nbs1)

• Assess ATM S1981 phosphorylation status after DNA damage using the Phospho-ATM (S1981) antibody

Research has demonstrated that functional MRN complex is required for efficient ATM activation after exposure to radiation or radiomimetic agents like neocarzinostatin . When the MRN complex is compromised, ATM S1981 phosphorylation is significantly reduced, indicating that the MRN complex likely serves as a sensor that recruits ATM to DNA damage sites and facilitates its autophosphorylation .

What are the essential controls when using Phospho-ATM (S1981) antibody in experimental studies?

When designing experiments with the Phospho-ATM (S1981) antibody, the following controls are essential for proper data interpretation:

Studies have shown that treatment with ATM inhibitors like Ku-55933 or wortmannin significantly reduces the phospho-S1981 signal, confirming that the detected phosphorylation is due to ATM kinase activity . Similarly, no phosphorylation signal is detected in ATM-deficient cells following irradiation, further validating antibody specificity .

How should samples be prepared to preserve ATM phosphorylation status?

Preserving ATM phosphorylation during sample preparation is critical for accurate results. The recommended protocol includes:

• Rapid sample processing to minimize dephosphorylation

• Inclusion of phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate, β-glycerophosphate) in all buffers

• Maintenance of cold temperatures during cell lysis and protein extraction

• Use of appropriate lysis buffers that effectively solubilize nuclear proteins

For Western blotting applications specifically:

• Process samples rapidly on ice

• Use PVDF membranes rather than nitrocellulose for better protein retention

• Consider reducing conditions and specific buffer compositions (e.g., Immunoblot Buffer Group 1) as demonstrated in validated protocols

For immunofluorescence applications:

• Fix cells promptly after treatment (typically with 4% paraformaldehyde)

• Include phosphatase inhibitors in washing buffers

• Optimize permeabilization conditions to maintain nuclear structure while allowing antibody access

What are common issues encountered when using Phospho-ATM (S1981) antibody and how can they be resolved?

Based on experimental evidence, phospho-ATM (S1981) appears as a high molecular weight band at approximately 350 kDa in Western blots . If this band is absent or if multiple bands appear, the experimental conditions should be carefully reassessed.

How can researchers distinguish between ATM S1981 phosphorylation and other phosphorylation events in the DNA damage response?

Distinguishing ATM S1981 phosphorylation from other phosphorylation events requires careful experimental design:

• Use multiple antibodies targeting different phospho-proteins in the DNA damage response (DDR) pathway

• Perform time-course experiments to establish temporal relationships

• Utilize ATM-specific inhibitors (e.g., Ku-55933) alongside broader PI3K inhibitors (e.g., wortmannin)

• Include ATM knockout/knockdown controls

A recommended panel of antibodies for comprehensive DDR analysis would include:

• Phospho-ATM (S1981)

• Phospho-ATR (Ser428)

• Phospho-DNA-PKcs (Ser2056)

• γH2AX (Ser139)

• Phospho-Chk2 (Thr68)

• Phospho-p53 (Ser15)

By comparing the phosphorylation patterns and their sensitivities to different inhibitors, researchers can determine which pathways are primarily responsible for the observed cellular responses. For example, ATM S1981 phosphorylation is particularly sensitive to Ku-55933, while ATR-mediated phosphorylation events are relatively resistant to this inhibitor .

How does ATM S1981 phosphorylation differ between various DNA damage types and cellular contexts?

The pattern and extent of ATM S1981 phosphorylation can vary significantly depending on the type of DNA damage and cellular context:

To investigate these differences, researchers should:

• Compare phosphorylation patterns after different damage types using the same antibody concentration and detection methods

• Assess colocalization with damage-specific markers

• Evaluate the requirement for the MRN complex across different damage types

• Analyze cell-type specific responses (cancer vs. normal cells, proliferating vs. quiescent)

Studies have shown that while DNA double-strand breaks strongly induce ATM S1981 phosphorylation through the MRN complex, other cellular stresses may activate ATM through different mechanisms, potentially resulting in different patterns or intensities of S1981 phosphorylation .

How can Phospho-ATM (S1981) antibody be used in multiplex immunofluorescence studies?

For advanced multiplex immunofluorescence studies examining ATM activation in relation to other DDR proteins:

• Select compatible antibodies raised in different host species or use directly conjugated primary antibodies

• Establish an optimized staining sequence that preserves epitope detection

• Include appropriate compensation controls for spectral overlap

• Utilize high-resolution confocal microscopy for co-localization analysis

A recommended multiplex panel for studying ATM activation at DNA damage sites:

Research has demonstrated that phospho-ATM (S1981) forms discrete nuclear foci that colocalize with γH2AX following DNA damage, providing a powerful visual readout of ATM activation at actual damage sites . The kinetics of this foci formation and resolution can provide insights into the efficiency of the DNA damage response in different experimental conditions.

How might Phospho-ATM (S1981) antibodies contribute to research on cancer therapies targeting the DNA damage response?

Phospho-ATM (S1981) antibodies offer significant potential for advancing cancer therapy research in several ways:

• Biomarker Development: The antibody can help identify tumors with altered ATM activation, potentially predicting responsiveness to treatments targeting the DNA damage response

• Therapy Response Monitoring: Measuring ATM S1981 phosphorylation before and after treatment can indicate therapy efficacy

• Combination Therapy Optimization: Evaluating how different therapeutic agents affect ATM activation can guide rational combination strategies

• Resistance Mechanism Investigation: Studying ATM phosphorylation in resistant tumors might reveal adaptation mechanisms

Methodological approaches for these applications include:

• Tissue microarray analysis of patient samples

• Ex vivo treatment of patient-derived organoids

• In vivo assessment of ATM activation in xenograft models

• Correlation of ATM phosphorylation with clinical outcomes

Given that ATM mutations are associated with ataxia-telangiectasia and increased cancer risk, and that ATM signaling critically influences cellular responses to radiation and chemotherapy, this antibody provides a valuable tool for translational research at the intersection of DNA damage, cancer biology, and therapeutic response .

What emerging technologies might enhance the application of Phospho-ATM (S1981) antibody in research?

Several emerging technologies promise to expand the utility of Phospho-ATM (S1981) antibodies:

For implementation, researchers should consider:

• Adapting antibody concentrations for each technology's requirements

• Developing appropriate controls specific to each platform

• Comparing results across technologies for comprehensive understanding

• Combining complementary approaches for multi-dimensional analysis

These technologies would provide unprecedented insights into how ATM phosphorylation orchestrates the complex cellular response to DNA damage at both population and single-cell levels.