Phospho-H2AFX (S139) Antibody

What is Phospho-H2AFX (S139) and why is it important in research?

Phospho-H2AFX (S139) is a phosphorylated form of histone H2AX at serine 139, which serves as a well-established marker of DNA double-strand breaks (DSBs). This phosphorylation event is crucial in the cellular response to DNA damage, as it initiates signaling cascades that promote repair processes and regulate cell cycle checkpoints . Its importance in research stems from its role as an early and sensitive indicator of DNA damage, making it invaluable for studying genotoxicity, cancer development, and efficacy of DNA-damaging therapeutic agents .

How does H2AFX phosphorylation relate to the DNA damage response pathway?

H2AFX phosphorylation at serine 139 occurs rapidly following DNA double-strand breaks. This modification plays a key role in the cellular response to DNA damage by recruiting repair factors to damage sites and amplifying the damage signal . The phosphorylated H2AFX serves as a platform for the assembly of DNA repair complexes and checkpoint proteins that collectively determine whether a cell will pause to repair damage or undergo apoptosis if damage is too extensive . This phosphorylation is required for checkpoint-mediated arrest of cell cycle progression in response to DNA damage and for efficient repair of DNA double-strand breaks .

What are the structural characteristics of Phospho-H2AFX (S139)?

Phospho-H2AFX (S139) is a post-translationally modified variant of histone H2AX, which replaces conventional H2A in a subset of nucleosomes . The most significant feature is the phosphorylation at serine 139 in the C-terminal region, often referred to as the gamma (γ) site. The calculated molecular weight of H2AX is approximately 15 kDa, though in SDS-PAGE it typically appears at around 17 kDa . H2AFX is localized primarily in the nucleus, particularly at chromosomal regions where DNA damage has occurred .

What types of Phospho-H2AFX (S139) antibodies are available and how should researchers choose?

Researchers have several options when selecting Phospho-H2AFX (S139) antibodies, including polyclonal and monoclonal variants. Polyclonal antibodies like CABP0099 offer high sensitivity and recognize multiple epitopes, which can be advantageous for certain applications . Monoclonal antibodies like CABP0687 or clone 3F2 provide higher specificity and consistency between batches . The selection should be based on the experimental application (Western blot, immunohistochemistry, immunofluorescence, ELISA), species reactivity requirements (human, mouse, rat), and detection method compatibility .

How can researchers validate the specificity of a Phospho-H2AFX (S139) antibody?

Validation of Phospho-H2AFX (S139) antibody specificity should include multiple approaches:

• Western blot analysis comparing untreated cells with cells exposed to DNA-damaging agents (e.g., UV radiation, staurosporine, or etoposide)

• Peptide competition assays using the phosphorylated and non-phosphorylated peptides

• Immunofluorescence showing characteristic nuclear foci formation after DNA damage induction

• Comparing reactivity across multiple cell lines and tissue types

• Validation in knockout/knockdown systems where possible

Successful validation should demonstrate the expected ~17 kDa band in Western blots that increases after DNA damage and nuclear foci in immunofluorescence applications .

What are the optimal protocols for detecting Phospho-H2AFX (S139) using Western blotting?

For optimal Western blot detection of Phospho-H2AFX (S139):

• Sample preparation: Extract proteins using buffers containing phosphatase inhibitors to preserve phosphorylation

• Gel electrophoresis: Use 15-18% gels to properly resolve the small H2AX protein

• Transfer: Short transfer times (60-90 minutes) at lower voltages often yield better results

• Blocking: 5% BSA in TBST is preferable to milk-based blockers which contain phosphatases

• Antibody dilution: Use the recommended dilution ratios (typically 1:500-1:1000 for primary antibodies)

• Positive controls: Include lysates from cells treated with DNA-damaging agents like UV radiation or etoposide

• Loading controls: Total H2AX or other stable nuclear proteins should be used to normalize phosphorylation levels

The procedure should be optimized for specific cell types and applications, as sensitivity can vary between antibody sources and sample types .

How should researchers optimize immunofluorescence protocols for visualizing γH2AX foci?

For optimal immunofluorescence visualization of γH2AX foci:

• Fixation: 4% paraformaldehyde for 10-15 minutes at room temperature preserves nuclear structure

• Permeabilization: 0.2-0.5% Triton X-100 for 5-10 minutes allows antibody access while maintaining nuclear morphology

• Blocking: 5-10% normal serum from the species of the secondary antibody for 1 hour

• Primary antibody: Use recommended dilutions (typically 1:50-1:200) and incubate overnight at 4°C

• Secondary antibody: Fluorophore-conjugated antibodies at 1:200-1:1000 for 1 hour at room temperature

• Counterstaining: DAPI for nuclear visualization; avoid propidium iodide which can mask γH2AX signal

• Mounting: Anti-fade mounting medium to prevent photobleaching during imaging

• Imaging: Confocal microscopy provides optimal resolution of individual foci

Researchers should include positive controls (cells treated with radiation or DNA-damaging agents) and negative controls (omission of primary antibody) to validate staining specificity .

What are the key considerations for quantifying Phospho-H2AFX (S139) levels using ELISA?

When quantifying Phospho-H2AFX (S139) using ELISA methods:

• Sample preparation: Cell lysates should be prepared with phosphatase inhibitors to preserve phosphorylation status

• Antibody selection: Use antibody pairs validated for ELISA applications, such as those in commercial kits

• Standardization: Include standard curves using recombinant phosphorylated H2AX or cell lysates with known phosphorylation levels

• Normalization: Measure total H2AX simultaneously to calculate the phosphorylated fraction, which provides more meaningful data than absolute phospho-H2AX levels alone

• Controls: Include both positive controls (DNA damage-induced samples) and negative controls

• Sensitivity: The detection limit should be established for each experimental system

• Signal development: Optimize incubation times for chromogenic substrates to maximize sensitivity while avoiding saturation

Human Phospho-H2AX (S139) and Total H2AX ELISA kits offer a convenient sandwich-based approach for semi-quantitative assessment of H2AX phosphorylation in various sample types .

How can researchers differentiate between apoptotic pan-nuclear H2AX phosphorylation and DNA damage-induced foci?

Differentiating between apoptotic pan-nuclear H2AX phosphorylation and DNA damage-induced foci requires careful analysis:

• Morphological assessment: DNA damage-induced foci appear as distinct nuclear puncta, while apoptotic cells show uniform, intense pan-nuclear staining

• Temporal analysis: Track the evolution of staining patterns over time; true DNA damage foci typically appear within minutes of damage and resolve within hours

• Co-staining: Use markers of apoptosis (cleaved caspase-3, PARP cleavage) alongside γH2AX

• Nuclear morphology: Apoptotic cells show characteristic nuclear condensation and fragmentation

• Quantitative assessment: Measure integrated nuclear intensity versus foci number and intensity

• Dose response: Analyze the relationship between treatment dose and staining pattern; apoptotic pan-nuclear staining typically appears at higher, cytotoxic doses

Understanding these distinctions is crucial for accurate interpretation of experimental results, particularly in studies evaluating genotoxicity or DNA-damaging therapeutics .

What are the most common technical issues when using Phospho-H2AFX (S139) antibodies and how can they be addressed?

Addressing these issues requires systematic troubleshooting and optimization for specific experimental conditions and cell types .

How should researchers interpret changes in Phospho-H2AFX (S139) levels across different experimental timepoints?

Interpreting temporal changes in Phospho-H2AFX (S139) levels requires understanding the kinetics of the DNA damage response:

• Initial response (minutes): Rapid phosphorylation occurs at damage sites; increasing γH2AX indicates active damage sensing

• Peak response (0.5-2 hours): Maximum phosphorylation; reflects the full activation of the DNA damage response

• Resolution phase (4-24 hours): Decreasing levels indicate successful repair; persistent high levels suggest repair defects or ongoing damage

• Long-term effects (>24 hours): Residual foci may indicate unrepaired or complex damage; complete resolution suggests successful repair

Researchers should consider:

• Cell cycle phase effects on phosphorylation levels

• Relationship between phosphorylation intensity and damage severity

• Correlation with other DNA damage markers

• Cell type-specific variations in response kinetics

Quantitative assessment across multiple timepoints provides the most informative picture of DNA damage induction and repair capacity .

How can researchers use Phospho-H2AFX (S139) to evaluate DNA repair capacity in cancer cells?

Evaluating DNA repair capacity in cancer cells using Phospho-H2AFX (S139):

• Kinetic analysis: Measure the rate of γH2AX foci formation and resolution after standardized damage to assess repair efficiency

• Dose-response relationships: Compare γH2AX induction across damage levels to identify repair thresholds

• Co-localization studies: Examine γH2AX spatial relationship with specific repair factors (e.g., 53BP1, RAD51, BRCA1) to determine which repair pathways are active

• Combination with repair inhibitors: Use specific DNA repair inhibitors to determine pathway dependencies

• Correlation with genomic instability markers: Link γH2AX patterns to chromosomal abnormalities

• Patient-derived samples: Compare γH2AX responses in tumor versus normal tissues to identify repair deficiencies

• Therapeutic response prediction: Use baseline and post-treatment γH2AX levels to predict sensitivity to DNA-damaging agents

This approach can identify cancer-specific DNA repair defects that might be exploited therapeutically through synthetic lethality approaches .

What are the most effective experimental designs for studying the relationship between Phospho-H2AFX (S139) and cell cycle checkpoints?

To effectively study the relationship between Phospho-H2AFX (S139) and cell cycle checkpoints:

• Synchronization protocols: Use methods like double thymidine block or serum starvation/release to obtain cell populations at specific cell cycle phases

• Multi-parameter flow cytometry: Combine γH2AX staining with DNA content analysis (propidium iodide) and cell cycle markers (e.g., cyclin B1, phospho-histone H3)

• Live-cell imaging: Use fluorescent reporters for both γH2AX and cell cycle phase to track individual cells over time

• Checkpoint inhibition: Compare γH2AX dynamics with and without specific checkpoint inhibitors (ATM/ATR/CHK1/CHK2 inhibitors)

• Genetic approaches: Use cells with defined mutations in checkpoint genes to assess their impact on γH2AX dynamics

• Time-course experiments: Sample at multiple timepoints after damage to correlate γH2AX changes with cell cycle progression

These approaches can reveal how γH2AX contributes to checkpoint activation and maintenance, and how checkpoint status affects DNA repair .

How can researchers integrate Phospho-H2AFX (S139) data with other DNA damage response markers for comprehensive pathway analysis?

For comprehensive pathway analysis integrating Phospho-H2AFX (S139) with other DNA damage response markers:

• Multiplex immunostaining: Simultaneously detect γH2AX alongside other DDR proteins (53BP1, MDC1, BRCA1, RAD51, etc.) to assess pathway activation and coordination

• Sequential chromatin immunoprecipitation (ChIP): Determine the temporal order of factor recruitment to damage sites

• Proximity ligation assays: Detect protein-protein interactions between γH2AX and other repair factors in situ

• Correlation analysis: Quantitatively compare γH2AX levels with other phosphorylation events in the DDR pathway

• Inhibitor studies: Systematically inhibit upstream kinases to determine regulatory relationships

• Genetic dependency approaches: Use CRISPR/siRNA to create pathway component knockouts/knockdowns and assess effects on γH2AX

• Computational modeling: Integrate quantitative γH2AX data with other DDR markers to model pathway dynamics

This multi-faceted approach provides insights into the temporal and spatial organization of the DNA damage response network and identifies critical nodes for potential therapeutic targeting .

How is Phospho-H2AFX (S139) being used as a biomarker in clinical research?

Phospho-H2AFX (S139) is emerging as an important biomarker in clinical research through several applications:

• Cancer diagnostics: Assessment of γH2AX levels in tumor biopsies can indicate endogenous DNA damage levels and genomic instability

• Treatment response monitoring: Changes in γH2AX after chemotherapy or radiation can predict treatment efficacy

• Personalized medicine: γH2AX response patterns may identify patients likely to benefit from DNA damage-based therapies

• Drug development: Quantification of γH2AX serves as a pharmacodynamic endpoint in clinical trials of DNA-damaging agents

• Radiation biodosimetry: γH2AX measurements in peripheral blood lymphocytes can estimate radiation exposure

• Cancer risk assessment: Elevated baseline γH2AX may indicate defective DNA repair and increased cancer susceptibility

Immunohistochemical detection of phosphorylated H2AX in tissue samples, such as breast cancer specimens, provides valuable information about the DNA damage status of the tumor microenvironment and may correlate with prognostic factors and treatment outcomes .

What are the methodological considerations for using Phospho-H2AFX (S139) antibodies in tissue microarrays and clinical samples?

When using Phospho-H2AFX (S139) antibodies in tissue microarrays and clinical samples:

• Fixation standardization: Consistent fixation protocols are essential as phospho-epitopes are sensitive to fixation variables

• Antigen retrieval optimization: Heat-induced epitope retrieval using basic buffer (pH 9.0-9.6) typically provides optimal results for phospho-H2AX detection

• Antibody validation: Extensive validation with positive and negative controls is critical for clinical applications

• Signal amplification: Consider using signal amplification methods for detecting low-level phosphorylation

• Automated staining: Use automated platforms for consistency in clinical settings

• Quantitative analysis: Implement digital pathology and image analysis for objective quantification

• Pre-analytical variables: Document and control for pre-analytical variables (time to fixation, fixation duration)

• Scoring systems: Develop and validate standardized scoring systems for phospho-H2AX positivity

For immunohistochemical applications, the recommended antibody dilution for phospho-H2AX detection in paraffin-embedded tissues is typically 1:50-1:200, and appropriate controls should always be included .

How does the measurement of Phospho-H2AFX (S139) contribute to understanding treatment resistance mechanisms?

Measurement of Phospho-H2AFX (S139) provides critical insights into treatment resistance mechanisms:

• Repair capacity assessment: Rapid resolution of γH2AX foci after treatment may indicate enhanced repair capacity and resistance

• Checkpoint adaptation: Persistent γH2AX with continued cell cycling suggests checkpoint adaptation and potential genomic instability

• Pathway rewiring: Altered patterns of γH2AX induction or resolution may reveal compensatory pathway activation

• Heterogeneity analysis: Single-cell assessment of γH2AX can identify resistant subpopulations within tumors

• Combination therapy rationale: γH2AX patterns after treatment can guide rational selection of sensitizing agents

• Acquired resistance mechanisms: Changes in γH2AX dynamics during treatment course may indicate evolving resistance mechanisms

• Cross-resistance prediction: γH2AX response to one agent may predict resistance to mechanistically related treatments

By characterizing DNA damage signaling and repair through γH2AX analysis, researchers can identify specific resistance mechanisms and develop strategies to overcome them, potentially through combination approaches targeting both DNA damage induction and repair inhibition .

What are the emerging technologies for high-throughput analysis of Phospho-H2AFX (S139) in research settings?

Emerging technologies for high-throughput analysis of Phospho-H2AFX (S139) include:

• Automated high-content imaging: Systems combining automated microscopy with sophisticated image analysis algorithms for rapid quantification of γH2AX foci

• Flow cytometry-based methods: Multi-parameter flow cytometry combining γH2AX with cell cycle markers and other DDR proteins

• Mass cytometry (CyTOF): Single-cell analysis of γH2AX alongside dozens of other cellular markers

• ELISA-based arrays: Parallel analysis of multiple samples in 96-well or 384-well formats

• Microfluidic platforms: Integration of cell culture, treatment, and γH2AX detection for rapid screening

• Single-cell sequencing combined with γH2AX immunoprecipitation: To map damage distribution across the genome

• Automated Western blot systems: For higher throughput protein analysis with reduced variability

These technologies enable screening of compound libraries for DNA-damaging effects, testing large patient cohorts, and performing detailed temporal analyses that were previously impractical .

How do different DNA damage types influence the pattern and intensity of H2AFX phosphorylation?

Different DNA damage types produce distinctive patterns of H2AFX phosphorylation:

• Ionizing radiation: Discrete nuclear foci corresponding to direct double-strand breaks, with number proportional to dose

• Replication stress: Smaller, more numerous foci associated with stalled replication forks, often S-phase specific

• UV radiation: Pan-nuclear γH2AX staining or smaller foci pattern reflecting nucleotide excision repair sites

• Chemical crosslinkers: Distinctive patterns of clustered foci related to inter-strand crosslinks

• Topoisomerase inhibitors: Large, bright foci at sites of trapped topoisomerase complexes

• Oxidative damage: Diffuse staining pattern with some discrete foci

• Oncogene-induced damage: Heterogeneous pattern reflecting replication stress and collapsed forks

Understanding these pattern differences allows researchers to infer the nature of DNA damage present in experimental or clinical samples and select appropriate analysis methods. Additionally, the intensity of phosphorylation can indicate the severity of damage and the cell's capacity to detect and respond to specific damage types .

What are the current limitations in Phospho-H2AFX (S139) research and how might they be addressed in future studies?

Current limitations in Phospho-H2AFX (S139) research include:

• Specificity challenges: Phospho-H2AFX can be induced by processes other than DNA damage, including certain stages of normal cell division

  • Solution: Combine with other damage markers and cellular context information

• Quantification standardization: Lack of standardized methods for quantifying γH2AX across laboratories

  • Solution: Develop reference standards and consensus quantification protocols

• Temporal resolution: Most analyses provide snapshots rather than continuous monitoring

  • Solution: Develop live-cell compatible γH2AX sensors with minimal interference

• Sensitivity limits: Detection of low-level, physiologically relevant damage can be challenging

  • Solution: Develop amplification methods that preserve spatial information

• Context dependence: γH2AX response varies with cell type, cell cycle, and microenvironment

  • Solution: Create comprehensive reference datasets across multiple conditions

• Functional significance: The presence of γH2AX doesn't always correlate with functional outcomes

  • Solution: Integrate with functional assays of cell survival, mutation frequency, etc.

• Technical variability: Antibody performance can vary between lots and manufacturers

  • Solution: Implement rigorous validation protocols and reporting standards

Future studies should aim to address these limitations through technological innovations, standardization efforts, and integration with other analytical approaches to provide a more complete understanding of DNA damage signaling dynamics .