H2AFX Antibody

What is H2AFX and why is it important in research?

H2AFX (H2A Histone Family Member X) is a variant of the histone H2A protein that becomes rapidly phosphorylated at serine 139 (forming γ-H2AX) in response to DNA double-strand breaks. This phosphorylation serves as a critical biomarker for DNA damage and repair processes . The importance of H2AFX in research stems from its role as an early and sensitive indicator of genotoxic stress, making it valuable for studying DNA damage mechanisms, cancer biology, drug development, and toxicology. Detection of γ-H2AX foci directly represents DNA double-strand break formation and allows researchers to quantify DNA damage in various experimental contexts .

What applications are H2AFX antibodies suitable for?

H2AFX antibodies are validated for multiple experimental applications depending on the specific antibody formulation. Common applications include:

When designing experiments, it's crucial to select an antibody that has been validated for your specific application and target species .

How should H2AFX antibodies be stored and handled?

For optimal performance, H2AFX antibodies should be stored according to manufacturer recommendations. Generally, long-term storage at -20°C is recommended for up to one year. For frequent use and short-term storage (up to one month), keeping the antibody at 4°C is acceptable . Repeated freeze-thaw cycles should be avoided as they can degrade the antibody and reduce its effectiveness. When handling the antibody, maintain sterile conditions and use appropriate pipetting techniques to prevent contamination. Always briefly centrifuge antibody vials before opening to collect the liquid at the bottom of the tube .

How should I select the appropriate H2AFX antibody for my experiment?

Selection of the appropriate H2AFX antibody depends on several critical factors:

• Epitope specificity: Determine whether you need antibodies recognizing total H2AFX or phosphorylated γ-H2AX (pSer139). Phospho-specific antibodies are crucial for DNA damage studies .

• Host species: Consider compatibility with other antibodies in multiplexed experiments. Rabbit monoclonal antibodies like M00241-1 offer high specificity and sensitivity .

• Clonality: Monoclonal antibodies provide consistent results with high specificity for a single epitope, while polyclonal antibodies recognize multiple epitopes and may offer higher sensitivity .

• Validated applications: Ensure the antibody has been validated for your specific application (WB, IHC, IF, etc.) and experimental conditions .

• Species reactivity: Verify cross-reactivity with your model organism. Some antibodies like M00241-1 react with human, mouse, and rat samples, while others may have more limited reactivity .

• Binding region: Some antibodies target specific amino acid regions, such as AA 130-142 or N-terminal versus C-terminal epitopes .

Review validation data and published literature using your antibody of interest to ensure it performs reliably in your experimental context.

What are the best positive and negative controls for H2AFX antibody experiments?

Implementing appropriate controls is essential for validating H2AFX antibody experiments:

Positive Controls:

• Cell lines treated with known DNA-damaging agents such as topotecan, etoposide, or ionizing radiation, which induce γ-H2AX formation

• A375 xenograft samples treated with topotecan (established model for γ-H2AX induction)

• Human breast hyperplasia or testis cancer tissue sections (demonstrated positive H2AFX staining)

Negative Controls:

• Untreated cell samples (baseline comparison)

• Cells treated with non-DNA damaging agents like rapamycin (which affects proliferation but not DNA damage)

• Primary antibody omission control

• Isotype-matched control antibody

• Blocking peptide competition assay to confirm specificity

Additionally, time-course experiments can serve as internal controls, as γ-H2AX levels typically peak 1-4 hours after damage induction and decrease as repair progresses .

What fixation method is optimal for detecting H2AFX in tissue samples?

The fixation method significantly impacts H2AFX antibody performance in tissue samples. Based on available data:

• Paraformaldehyde (PFA) is recommended for H2AFX detection due to its superior tissue penetration properties. Importantly, PFA should be freshly prepared before use, as long-term stored PFA converts to formalin as the molecules aggregate .

• Heat-mediated antigen retrieval in EDTA buffer (pH 8.0) has been successfully used for paraffin-embedded sections when working with anti-H2AFX antibodies. This process helps expose epitopes that may be masked during fixation .

• Blocking conditions: 10% goat serum has been effectively used to block non-specific binding sites before antibody incubation .

For optimal immunohistochemical detection, tissue sections should be incubated with primary antibody (e.g., 1:200 dilution of rabbit anti-H2AFX) overnight at 4°C, followed by appropriate secondary antibody incubation (e.g., Peroxidase Conjugated Goat Anti-rabbit IgG) for 30 minutes at 37°C .

What are the different methods for quantifying γ-H2AX and their relative advantages?

Multiple methods exist for γ-H2AX quantification, each with distinct advantages and limitations:

The choice of method depends on research goals: foci counting provides detailed spatial information but is labor-intensive, while DELFIA offers superior sensitivity for detecting low levels of DNA damage (femtogram range) .

How can I optimize immunofluorescence detection of γ-H2AX foci?

Optimizing immunofluorescence detection of γ-H2AX foci requires attention to several parameters:

• Fixation protocol: Standard protocol includes 4% paraformaldehyde fixation for 10-15 minutes at room temperature, which preserves nuclear architecture while maintaining antigen accessibility .

• Permeabilization: Use 0.2-0.5% Triton X-100 in PBS to ensure antibody access to nuclear antigens while minimizing background.

• Blocking conditions: 5-10% serum (matching the host of the secondary antibody) effectively blocks non-specific binding sites .

• Antibody dilution optimization: Titrate primary and secondary antibodies to determine optimal concentrations that maximize specific signal while minimizing background. A 1:200 dilution has been effective for some H2AFX antibodies .

• Incubation time and temperature: Overnight incubation at 4°C for primary antibody often yields best results, followed by 30-60 minute room temperature incubation for secondary antibody .

• Counterstaining: DAPI or Hoechst nuclear counterstain helps define nuclear boundaries for accurate foci quantification.

• Image acquisition settings: Use consistent exposure times, gain, and offset settings across experimental conditions. Z-stack imaging can improve foci detection within the nuclear volume.

These parameters should be systematically optimized for each experimental system and antibody to ensure reliable and reproducible results.

What is the significance of different phosphorylation sites on H2AFX?

H2AFX undergoes various post-translational modifications, with phosphorylation being particularly important:

• Serine 139 (pSer139): The most extensively studied phosphorylation site, forming γ-H2AX in response to DNA double-strand breaks. This modification serves as a critical biomarker for DNA damage and repair processes . Antibodies specific to this phosphorylation site are commonly used in DNA damage research.

• Serine 140 (pSer140): Another phosphorylation site with distinct biological functions. Antibodies targeting pSer140 are available for applications including WB, ELISA, IHC, ICC, FACS, and IF .

• Lysine 119 ubiquitination (ubLys119): This modification plays roles in chromatin regulation and DNA repair pathways. Specific antibodies can detect this ubiquitination .

The choice of which modification to study depends on the research question:

• For general DNA damage assessment, pSer139 (γ-H2AX) antibodies are most commonly used

• For mechanistic studies of histone regulation, antibodies targeting other modifications may provide additional insights

• For comprehensive analysis, using multiple antibodies targeting different modifications can reveal the interplay between different signaling pathways

Each modification represents distinct biological processes and may respond differently to various experimental treatments.

How can H2AFX antibodies be used for pharmacodynamic studies of DNA-damaging agents?

H2AFX antibodies provide powerful tools for pharmacodynamic assessment of DNA-damaging therapeutic agents. A validated approach includes:

• Experimental design: Collect biopsies or samples at strategic timepoints after drug administration (e.g., 1, 2, 4, and 7 hours) using clinically relevant dosing regimens. This allows determination of both the magnitude and kinetics of DNA damage response .

• Dose-response relationship: Test multiple drug concentrations (e.g., 0.03, 0.1, and 0.32 MTD) to establish dose-dependent γ-H2AX response curves, essential for determining minimal effective doses .

• Quantification method: Employ validated immunofluorescence assays with novel data quantitation and image processing algorithms to determine nuclear-specific γ-H2AX staining .

• Sample types: Use tumor needle biopsies from xenograft models or clinical samples. Additional accessible tissues like hair follicles can serve as surrogate markers in clinical settings .

• Reference compounds: Include known DNA-damaging agents (e.g., topotecan for Top1 inhibitors) as positive controls and non-DNA damaging agents (e.g., rapamycin) as negative controls to validate assay specificity .

This approach has been successfully employed to compare pharmacodynamic properties of structurally related topoisomerase I inhibitors and provides a framework for evaluating novel therapeutic compounds in both preclinical and clinical settings .

What are the challenges in interpreting γ-H2AX data from heterogeneous tissue samples?

Interpreting γ-H2AX data from heterogeneous tissue samples presents several challenges that researchers must address:

• Cellular heterogeneity: Tissues contain multiple cell types with varying baseline levels of γ-H2AX and differential responses to treatments. Researchers should consider:

  • Cell type-specific γ-H2AX patterns

  • Varying proliferation rates affecting background γ-H2AX levels

  • Differential drug penetration across the tissue

• Technical variability: Inconsistent fixation, antibody penetration, and staining efficiency across the tissue can create artifacts. Standardized protocols and internal controls are essential .

• Quantification approaches: Several strategies can address heterogeneity:

  • Cell type-specific markers for co-localization analysis

  • Stratified analysis of different tissue regions

  • Single-cell analysis to capture population distribution rather than averages

  • Automated imaging platforms with machine learning algorithms to identify specific cell populations

• Reference standards: Include internal positive controls (e.g., irradiated tissue sections) and negative controls processed alongside experimental samples to normalize for technical variation .

• Biological interpretation: Consider that γ-H2AX may indicate not only DNA damage but also normal biological processes such as recombination, senescence, or apoptosis, requiring careful correlation with additional biological markers.

Addressing these challenges requires combining advanced imaging techniques, sophisticated quantification algorithms, and thoughtful experimental design to extract meaningful biological insights from complex tissue samples.

How do different antibody clones affect the detection of H2AFX in various experimental contexts?

The choice of antibody clone can significantly impact H2AFX detection outcomes across different experimental contexts:

• Epitope specificity: Different clones recognize distinct epitopes on H2AFX. Some target total H2AFX protein (e.g., AA 130-142, N-terminal, or C-terminal regions), while others specifically detect post-translational modifications like phosphorylation at Ser139 (γ-H2AX) . This epitope specificity determines what biological state of H2AFX is being detected.

• Application performance: Clones vary in their performance across applications:

  • Some clones work exceptionally well for western blotting but poorly for immunohistochemistry

  • Others may be optimized for immunofluorescence but less effective for flow cytometry

  • Validation data should be consulted for each specific application

• Cross-reactivity: Antibody clones differ in their species cross-reactivity profiles. Some recognize human H2AFX only, while others (like M00241-1) cross-react with mouse and rat, enabling consistent reagent use across multiple model systems .

• Background and specificity issues: Different clones can exhibit variable non-specific binding patterns:

  • Monoclonal antibodies typically offer higher specificity but may be sensitive to epitope masking

  • Polyclonal antibodies recognize multiple epitopes, potentially improving detection but sometimes at the cost of increased background

• Sensitivity to fixation and sample preparation: Some clones may be particularly sensitive to fixation conditions, working well with PFA but poorly with formalin, or requiring specific antigen retrieval methods .

Researchers should select antibody clones based on published validation data for their specific application and conduct preliminary validation experiments comparing multiple clones when establishing new assays or working with challenging sample types.

What are common issues encountered when using H2AFX antibodies and how can they be resolved?

Researchers frequently encounter several challenges when working with H2AFX antibodies. Here are common issues and their solutions:

For immunohistochemistry applications specifically, optimizing heat-mediated antigen retrieval in EDTA buffer, using fresh PFA for fixation, and implementing appropriate blocking with 10% serum can significantly improve results .

How can I validate the specificity of H2AFX antibodies for my experimental system?

Validating H2AFX antibody specificity is crucial for experimental reliability. A comprehensive validation approach includes:

• Positive and negative controls:

  • Treat cells with known DNA-damaging agents (e.g., topotecan, etoposide) as positive controls

  • Include undamaged cells as negative controls

  • Use siRNA knockdown or CRISPR knockout of H2AFX to confirm specificity

• Competing peptide assay: Pre-incubate the antibody with the immunizing peptide (e.g., synthetic peptide derived from human Histone H2AX for M00241-1) to block specific binding .

• Comparative antibody testing: Test multiple antibodies targeting different epitopes of H2AFX and compare staining patterns.

• Western blot validation: Confirm antibody detects a band of the expected molecular weight (~15 kDa for H2AFX).

• Correlation with other DNA damage markers: Verify co-localization with other DNA damage markers like 53BP1, MDC1, or DNA-PKcs.

• Dose-response relationship: Establish that γ-H2AX signal increases proportionally with increasing doses of DNA-damaging agents .

• Analysis across sample types: Verify consistent staining patterns across different sample preparations (cultured cells, fresh tissue, paraffin-embedded tissue) .

• Time-course analysis: Confirm that γ-H2AX kinetics follow expected patterns (rapid increase after damage, gradual decrease during repair) .

Documentation of these validation steps should be maintained as part of laboratory protocols to ensure ongoing experimental quality.

What are the best approaches for multiplex detection of H2AFX with other DNA damage markers?

Multiplex detection of H2AFX with other DNA damage markers provides comprehensive insights into DNA damage response mechanisms. Optimal approaches include:

• Compatible antibody selection:

  • Choose primary antibodies raised in different host species (e.g., rabbit anti-H2AFX with mouse anti-53BP1)

  • If antibodies are from the same host, use directly conjugated primary antibodies or sequential immunostaining protocols

  • Verify that epitope retrieval conditions are compatible for all target proteins

• Fluorophore selection for immunofluorescence:

  • Select fluorophores with minimal spectral overlap (e.g., Alexa Fluor 488, 555, 647)

  • Consider brightness and photostability when matching fluorophores to target abundance

  • Include single-color controls to verify specificity and set compensation parameters

• Advanced detection methods:

  • DELFIA (dissociation-enhanced lanthanide fluorescence immunoassay) offers superior sensitivity (nanomolar resolution) for detecting low levels of γ-H2AX

  • Time-resolved fluorescence can reduce background and improve detection sensitivity

  • Mass cytometry (CyTOF) allows highly multiplexed detection without spectral overlap

• Imaging and analysis strategies:

  • Confocal microscopy with z-stacking for 3D colocalization analysis

  • Super-resolution microscopy (STORM, PALM, SIM) for detailed spatial relationships

  • Automated image analysis algorithms that quantify colocalization coefficients and spatial relationships

• Validation approaches:

  • Confirm expected temporal relationships (some markers appear earlier than others)

  • Verify biological relationships (e.g., γ-H2AX should colocalize with 53BP1 at DNA damage sites)

  • Include appropriate controls for each marker in the multiplex panel

These approaches allow researchers to simultaneously assess multiple aspects of the DNA damage response, providing deeper insights into complex biological processes.