H2AFX (Ab-139) Antibody

What is H2AFX (Ab-139) Antibody and what does it specifically detect?

H2AFX (Ab-139) Antibody specifically recognizes histone H2AX phosphorylated at serine 139, commonly referred to as gamma-H2AX (γ-H2AX). This antibody targets the peptide sequence around serine 139 of human histone H2AX . The phosphorylation of H2AX at this site occurs rapidly in response to DNA double-strand breaks (DSBs), making it a highly sensitive marker for DNA damage detection .

The antibody is available in both polyclonal and monoclonal formats:

• Polyclonal antibodies (typically rabbit-derived) offer broad epitope recognition

• Monoclonal antibodies (like clone 3F2) provide highly specific detection with reduced background

This antibody serves as an essential tool for researchers studying DNA damage response mechanisms, cancer biology, genotoxicity, and cellular stress responses .

What applications is H2AFX (Ab-139) Antibody validated for?

H2AFX (Ab-139) Antibody has been validated for multiple research applications across different platforms:

When selecting appropriate dilutions, it's advisable to perform a titration experiment with your specific cell type and treatment conditions to optimize signal-to-noise ratio .

How should H2AFX (Ab-139) Antibody be stored and handled for optimal performance?

Proper storage and handling are critical for maintaining antibody performance:

• Temperature: Store at -20°C for long-term preservation, with minimal freeze-thaw cycles. Short-term storage at 4°C is acceptable for up to 2 weeks

• Buffer composition: Most commercial preparations are supplied in buffered solutions containing:

  • 50% glycerol

  • Phosphate-buffered saline (PBS) at pH 7.4

  • 0.02-0.03% preservative (typically Proclin 300 or sodium azide)

• Aliquoting: Divide into small working aliquots upon receipt to avoid repeated freeze-thaw cycles

• Handling precautions:

  • Avoid contamination

  • Centrifuge briefly before opening

  • Allow to equilibrate to room temperature before use

These storage conditions ensure antibody stability and consistent performance across multiple experiments.

What are the optimal protocols for detecting γ-H2AX foci using H2AFX (Ab-139) Antibody in immunofluorescence?

For optimal γ-H2AX foci detection by immunofluorescence, follow these key protocol steps:

Cell preparation and fixation:

• Grow cells on coverslips or chamber slides to appropriate density

• Fix cells with either 100% methanol (5 min) or 4% paraformaldehyde (10-15 min)

• Permeabilize with 0.1% PBS-Triton X-100 for 5 minutes

Blocking and antibody incubation:

• Block with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1 hour

• Incubate with primary H2AFX (Ab-139) antibody (1:50-1:200 dilution) overnight at 4°C

• Wash thoroughly with PBS (3 × 5 minutes)

• Incubate with fluorophore-conjugated secondary antibody (e.g., Alexa Fluor 488) at 1:1000 dilution for 1 hour at room temperature

• Counterstain nuclei with DAPI

Mounting and imaging:

• Mount slides with anti-fade mounting medium

• Image using a confocal or epifluorescence microscope with appropriate filters

• For optimal foci detection, use a 63× or 100× oil immersion objective

Key considerations:

• Include appropriate positive controls (e.g., cells treated with etoposide or ionizing radiation)

• Include negative controls (omitting primary antibody)

• For co-staining experiments, ensure compatible primary antibodies from different host species

This protocol has been validated in multiple experimental systems and provides clear visualization of discrete nuclear γ-H2AX foci .

How can I quantify γ-H2AX signal in my experiments?

Several methods exist for quantifying γ-H2AX, each with specific advantages depending on your research question:

1. Manual foci counting (traditional approach):

• Count discrete nuclear foci in individual cells using fluorescence microscopy

• Typically 50-100 cells are counted per condition

• Advantages: Direct visualization of foci morphology and nuclear distribution

• Limitations: Time-consuming, potential for subjective bias, difficult with overlapping foci

2. Automated image analysis:

• Use software like BioTek Spot Count algorithm or CellProfiler

• Parameters to measure: foci number, size, intensity, and nuclear distribution

• Advantages: Higher throughput, reduced subjective bias

• Limitations: May have errors with overlapping or clustered foci

3. Flow cytometry:

• Measure total cell fluorescence intensity

• Enables analysis of thousands of cells rapidly

• Advantages: Statistical robustness, cell cycle correlation possible

• Limitations: Loses spatial information about foci distribution

4. Dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA):

• Time-resolved fluorescence detection using europium-tagged secondary antibodies

• Advantages: Nanomolar sensitivity, superior signal-to-background ratio, quantitative measurement

• Limitations: Specialized equipment required

5. Western blotting:

• Quantify total γ-H2AX protein levels

• Advantages: Simple to perform, good for global response

• Limitations: Loses single-cell and spatial information

When selecting a quantification method, consider your specific research question, available equipment, and desired throughput .

What controls should be included when using H2AFX (Ab-139) Antibody?

Proper experimental controls are essential for reliable interpretation of γ-H2AX data:

Positive controls:

• Cells treated with known DNA damaging agents:

  • Etoposide (1-100 μM, 1-1.5 hours) - topoisomerase II inhibitor

  • Ionizing radiation (2-10 Gy)

  • Staurosporine - induces apoptosis-related DNA fragmentation

Negative controls:

• Untreated cells (baseline control)

• Primary antibody omission (to assess secondary antibody specificity)

• Isotype control antibody (to assess non-specific binding)

Additional recommended controls:

• Phosphatase treatment of samples (should eliminate γ-H2AX signal)

• Cell cycle synchronization controls (S-phase cells may show constitutive γ-H2AX)

• Loading controls for Western blots (e.g., GAPDH, total H2AX, or α-tubulin)

For phospho-specific validation:

• Compare treated versus untreated samples

• Include a phospho-blocking peptide control

• Consider analyzing signal in phosphatase-treated samples

Including these controls will enhance the reliability and interpretability of your γ-H2AX data across different experimental platforms.

How can I distinguish between DNA damage-induced γ-H2AX and constitutive H2AX phosphorylation during the cell cycle?

Distinguishing between DNA damage-induced and constitutive γ-H2AX is critical for accurate data interpretation:

Characteristics of constitutive γ-H2AX:

• Predominantly occurs in S and G2M phases of the cell cycle

• Lower intensity compared to damage-induced foci

• More diffuse staining pattern

• Present even in untreated cells

• Varies among cell lines and can be influenced by p53 status

Methods for differentiation:

• Cell cycle analysis correlation:

  • Perform dual staining for γ-H2AX and DNA content (propidium iodide)

  • Analyze by flow cytometry to correlate γ-H2AX signal with cell cycle phase

  • G1 cells have minimal constitutive γ-H2AX

• Reactive oxygen species (ROS) scavenger treatment:

  • Treat cells with N-acetyl-L-cysteine (NAC, 1-10 mM)

  • NAC reduces constitutive γ-H2AX by neutralizing ROS

  • DNA damage-induced γ-H2AX is less affected by NAC treatment

• Morphological discrimination:

  • DNA damage-induced foci: Discrete, bright, larger foci

  • Constitutive γ-H2AX: More diffuse, smaller, and less intense

  • Quantify using high-resolution microscopy

• Kinetics analysis:

  • DNA damage-induced γ-H2AX shows rapid induction and slower resolution

  • Track foci formation and dissolution over time (30 min - 24 hrs)

Research has shown that constitutive H2AX phosphorylation is markedly higher in cells with wild-type p53 compared to p53-deficient or mutant cells across all cell cycle phases, suggesting p53 may facilitate H2AX phosphorylation .

What factors affect γ-H2AX detection and how can I optimize sensitivity?

Multiple factors can influence γ-H2AX detection sensitivity and should be optimized:

Biological factors:

• Cell type: Different cell lines show variable baseline γ-H2AX levels

• Cell cycle phase: S and G2M cells have higher constitutive γ-H2AX levels

• p53 status: Wild-type p53 cells show higher constitutive γ-H2AX

• Growth conditions: Confluency and serum levels affect ROS generation

Technical factors:

• Fixation method:

  • Methanol fixation may preserve nuclear structure better than paraformaldehyde

  • Optimal fixation time is critical (typically 5-15 minutes)

• Antibody selection:

  • Monoclonal antibodies (e.g., clone 3F2) may provide higher specificity

  • Polyclonal antibodies might offer better signal amplification

  • Validation with multiple antibody clones is recommended for critical experiments

• Detection method optimization:

  • Microscopy: Use confocal over widefield for better spatial resolution

  • Flow cytometry: Adjust compensation and gating strategies

  • DELFIA: Optimize europium-chelate concentration and incubation times

• Signal amplification strategies:

  • Tyramide signal amplification for low-level detection

  • Enhanced chemiluminescence for Western blotting

  • Biotin-streptavidin systems for immunohistochemistry

Sensitivity enhancement approaches:

• Pre-extraction of soluble proteins before fixation enhances foci visualization

• Antigen retrieval (citrate buffer, pH 6.0) for formalin-fixed tissues

• Signal-to-noise ratio optimization by titrating primary and secondary antibodies

These optimizations are particularly important when studying low levels of DNA damage or when examining subtle differences between experimental conditions.

How does γ-H2AX signal intensity correlate with the extent of DNA damage?

The relationship between γ-H2AX signal and DNA damage is complex but follows several established patterns:

Quantitative relationship:

• Each DNA double-strand break (DSB) typically generates a discrete γ-H2AX focus

• One γ-H2AX focus is estimated to correspond to one DSB

• Linear correlation exists between radiation dose and γ-H2AX foci number at low damage levels

• At high damage levels, foci may overlap and become difficult to distinguish individually

Signal dynamics and interpretation:

Research findings on correlation:

• Studies show strong correlation between γ-H2AX foci and radiation dose (0.1-2 Gy)

• Etoposide concentration (0.01-100 μM) shows dose-dependent γ-H2AX induction

• γ-H2AX is more sensitive than other DNA damage markers, detecting damage at nanomolar concentrations of genotoxic agents

For accurate interpretation, researchers should consider both γ-H2AX focus number and intensity characteristics, while also accounting for confounding factors like cell cycle phase and constitutive phosphorylation .

What are the considerations for using H2AFX (Ab-139) Antibody in combination with other DNA damage markers?

Multiplexed detection of γ-H2AX with other DNA damage response proteins provides more comprehensive insight into damage response mechanisms:

Compatible marker combinations:

• DNA damage sensors and mediators:

  • 53BP1 (p53-binding protein 1) - co-localizes with γ-H2AX at DSB sites

  • MDC1 (mediator of DNA damage checkpoint 1) - directly binds γ-H2AX

  • ATM (phosphorylated) - kinase responsible for H2AX phosphorylation

• DNA repair pathway markers:

  • RAD51 - homologous recombination

  • Ku70/80 - non-homologous end joining

  • BRCA1/BRCA2 - homologous recombination regulators

• Cell cycle markers:

  • Cyclin A or PCNA - S-phase markers

  • Phospho-histone H3 - mitotic marker

  • p21 - cell cycle arrest indicator

Technical considerations:

• Antibody compatibility:

  • Use primary antibodies from different host species

  • If using same-species antibodies, consider sequential staining with blocking steps

  • Test for cross-reactivity between secondary antibodies

• Fluorophore selection:

  • Choose spectrally distinct fluorophores to minimize bleed-through

  • Consider signal strength (γ-H2AX often gives strong signal and can use dim fluorophores)

  • Account for cellular autofluorescence when selecting fluorophores

• Quantification strategies:

  • Co-localization analysis requires high-resolution imaging

  • Calculate Pearson's or Mander's coefficient for co-localization quantification

  • Consider 3D analysis for accurate spatial relationships

Experimental protocol example:
The following protocol has been validated for dual staining of γ-H2AX and α-tubulin:

• Fix cells with 100% methanol (5 min)

• Permeabilize with 0.1% PBS-Triton X-100 (5 min)

• Block with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween (1 hr)

• Co-incubate with H2AFX (Ab-139) antibody (rabbit, 0.1 μg/ml) and Anti-alpha Tubulin (mouse, clone DM1A)

• Wash and incubate with species-specific secondary antibodies:

  • Goat Anti-Rabbit IgG (Alexa Fluor 488)

  • Goat Anti-Mouse IgG (Alexa Fluor 594)

• Counterstain nuclei with DAPI

This multiplexed approach enables correlation between DNA damage, cellular structures, and cell cycle status within individual cells.

How can H2AFX (Ab-139) Antibody be used to assess genotoxicity of compounds?

H2AFX (Ab-139) Antibody serves as a sensitive tool for genotoxicity assessment:

Advantages for genotoxicity testing:

• Highly sensitive detection of DNA damage

• Suitable for high-throughput screening

• Requires smaller sample volumes than traditional genotoxicity assays

• Can be used with various cell types including primary cells

• Detects damage at lower concentrations than comet assay or micronucleus test

Experimental approaches:

• Dose-response assessment:

  • Treat cells with increasing concentrations of test compound

  • Measure γ-H2AX induction using flow cytometry or high-content imaging

  • Determine lowest observable effect concentration

  • Example: Etoposide shows linear dose-response between 0.01-100 μM

• Time-course analysis:

  • Evaluate both induction and resolution of γ-H2AX signal

  • Persistent γ-H2AX indicates inability to repair damage

  • Typical timepoints: 30 min, 1 hr, 4 hr, 24 hr post-exposure

• High-throughput screening workflow:

  • Seed cells in 96-well plates

  • Treat with compound library at multiple concentrations

  • Fix and immunostain for γ-H2AX

  • Analyze using automated microscopy or plate-based cytometry

  • Include positive controls (etoposide or radiation) and negative controls

Quantification methods optimized for genotoxicity assessment:

• DELFIA assay: Provides nanomolar sensitivity, suitable for low-damage detection

• High-content imaging: Enables multi-parameter toxicity assessment

• Flow cytometry: Allows correlation with cell cycle and viability

The superior sensitivity of γ-H2AX detection makes it particularly valuable for identifying compounds with weak genotoxic potential that might be missed by traditional assays, though results should be confirmed with complementary methods for regulatory submissions .

What are the considerations for using H2AFX (Ab-139) Antibody in different tissue and cell types?

Different tissue and cell types require specific optimization strategies:

Cell line-specific considerations:

• Cancer cell lines:

  • Often have higher baseline γ-H2AX due to genomic instability

  • p53 status influences constitutive γ-H2AX levels

  • Examples: A549, HeLa, and TK6 cells are well-characterized for γ-H2AX studies

• Primary cells:

  • Generally lower background γ-H2AX than cancer cells

  • May require more sensitive detection methods

  • More physiologically relevant but potentially more variable

• Stem cells:

  • Enhanced DNA damage response mechanisms

  • Cell density and differentiation state affect γ-H2AX levels

  • Consider co-staining with stemness markers

Tissue-specific optimization:

• Formalin-fixed paraffin-embedded (FFPE) tissues:

  • Require antigen retrieval (heat-mediated using citrate buffer, pH 6.0)

  • Typical antibody dilution: 1:20-1:200

  • Background autofluorescence can be problematic (consider using chromogenic detection)

• Frozen tissues:

  • Better antigen preservation but poorer morphology

  • Fixation with acetone or methanol is often sufficient

  • Lower antibody concentrations typically needed

Optimization parameters by tissue/cell type:

When working with new cell or tissue types, preliminary experiments comparing different fixation methods, antibody dilutions, and detection systems are strongly recommended .

How can I analyze γ-H2AX in correlation with cell cycle phases?

Cell cycle correlation provides critical context for interpreting γ-H2AX data:

Methodological approaches:

• Flow cytometry dual parameter analysis:

  • Stain cells with γ-H2AX antibody and DNA content marker (propidium iodide or DAPI)

  • Gate cell populations by DNA content (G1, S, G2/M)

  • Analyze γ-H2AX intensity within each cell cycle phase

  • Typical dilution for flow cytometry: 1:200

• Immunofluorescence with cell cycle markers:

  • Co-stain with γ-H2AX and cell cycle-specific proteins:

    • PCNA or EdU incorporation (S-phase)

    • Cyclin D1 (G1)

    • Phospho-histone H3 (mitosis)

  • Analyze co-localization or mutual exclusivity patterns

• Cell synchronization approaches:

  • Synchronize cells using methods like double thymidine block

  • Collect at defined timepoints after release

  • Analyze γ-H2AX in populations enriched for specific cell cycle phases

Interpreting cell cycle-specific γ-H2AX patterns:

• G1 phase:

  • Lowest constitutive γ-H2AX levels

  • Clearest distinction between damaged and undamaged cells

  • Ideal phase for detecting exogenous damage

• S phase:

  • Elevated constitutive γ-H2AX due to replication stress

  • More diffuse staining pattern

  • May reflect stalled replication forks rather than DSBs

• G2/M phase:

  • Intermediate γ-H2AX levels

  • Signal may reflect unrepaired damage persisting from S phase

  • Cell cycle checkpoints may arrest cells with high γ-H2AX

Research findings on cell cycle correlation:
Studies have shown that constitutive γ-H2AX levels vary significantly between cell cycle phases, with S and G2M cells showing 2-3 fold higher γ-H2AX expression compared to G1 cells. This pattern has been observed across multiple cell lines including TK6, WTK1, NH32, and HL-60, though the absolute levels differ based on p53 status .

The application of ROS scavengers like N-acetyl-L-cysteine (NAC) reduces constitutive γ-H2AX most prominently in S-phase cells, suggesting ROS-induced DNA damage during replication contributes to baseline γ-H2AX levels .

Understanding these cell cycle variations is essential for properly interpreting γ-H2AX data, particularly in heterogeneous cell populations.

What are common issues encountered with H2AFX (Ab-139) Antibody and how can they be resolved?

Researchers may encounter several challenges when working with H2AFX (Ab-139) Antibody:

Problem: High background signal

• Possible causes:

  • Insufficient blocking

  • Excessive antibody concentration

  • Non-specific binding

  • Cell autofluorescence

• Solutions:

  • Increase blocking time (2-3 hours) or concentration (3-5% BSA)

  • Optimize antibody dilution through titration experiments

  • Include 0.1-0.3M glycine in blocking buffer to reduce aldehyde-mediated background

  • Use different blocking agents (milk, serum, commercial blockers)

Problem: Weak or absent signal

• Possible causes:

  • Insufficient antigen retrieval (for FFPE samples)

  • Overfixation

  • Antibody degradation

  • Low level of target protein

• Solutions:

  • Optimize antigen retrieval (try citrate buffer pH 6.0)

  • Reduce fixation time

  • Use fresh antibody aliquots

  • Include positive controls (etoposide-treated cells)

  • Consider signal amplification systems

Problem: Non-specific bands in Western blot

• Possible causes:

  • Cross-reactivity with other phosphorylated proteins

  • Sample degradation

  • Secondary antibody issues

• Solutions:

  • Increase antibody dilution (1:1000-1:5000)

  • Add phosphatase inhibitors to sample buffer

  • Optimize blocking and washing conditions

  • Test different membrane types (PVDF vs nitrocellulose)

Problem: Inconsistent foci counting results

• Possible causes:

  • Subjective threshold setting

  • Overlapping or clustered foci

  • Z-stack imaging issues

  • Cell cycle variability

• Solutions:

  • Use automated analysis with consistent parameters

  • Apply 3D analysis for overlapping foci

  • Standardize focal plane or use maximum intensity projections

  • Consider cell cycle synchronization or co-staining

Problem: High variability between experiments

• Possible causes:

  • Cell culture conditions affecting baseline ROS

  • Cell density differences

  • Variable fixation/permeabilization

  • Inconsistent imaging parameters

• Solutions:

  • Standardize cell culture conditions and passage number

  • Control cell density at seeding

  • Create detailed SOPs for sample processing

  • Use internal calibration standards in each experiment

Implementing these troubleshooting approaches can significantly improve the reliability and reproducibility of γ-H2AX detection using H2AFX (Ab-139) Antibody across different experimental systems.

How should γ-H2AX data be properly interpreted in the context of different DNA damage mechanisms?

Correct interpretation of γ-H2AX data requires understanding the relationship between signal patterns and specific DNA damage mechanisms:

Damage-specific γ-H2AX patterns:

• Ionizing radiation-induced damage:

  • Discrete, clearly defined nuclear foci

  • Random distribution throughout nucleus

  • Each focus typically represents one DSB

  • Linear dose-response at low doses (0.1-2 Gy)

  • Resolution kinetics: 50% reduction in 2-5 hours

• Replication stress:

  • Smaller, more numerous foci

  • Often co-localized with replication factories

  • S-phase specific

  • Examples: hydroxyurea, aphidicolin treatment

  • Often accompanied by ATR rather than ATM activation

• Topoisomerase inhibitors:

  • Etoposide (topo II): Discrete foci similar to radiation

  • Camptothecin (topo I): More diffuse pattern, S-phase dependent

  • Dose-dependent response observed with etoposide (0.01-100 μM)

• Apoptosis-related damage:

  • Pan-nuclear staining rather than distinct foci

  • Often accompanied by chromatin condensation

  • May show extremely bright signal

  • Distinct from repair-associated γ-H2AX patterns

• Oxidative damage:

  • Reduced by antioxidant treatment (e.g., NAC)

  • Often presents as constitutive phosphorylation

  • Higher in S and G2M phases

  • Cell type dependent, influenced by p53 status

Interpretation guidelines:

• Focus morphology analysis:

  • Small, numerous foci: Often replication-associated damage

  • Large, bright foci: Direct DSBs from clastogens

  • Pan-nuclear staining: Consider apoptosis or high damage load

  • Peripheral nuclear staining: Potential nuclease activity during apoptosis

• Temporal dynamics assessment:

  • Early appearance (<30 min): Direct DSB formation

  • Delayed appearance: Secondary damage or repair intermediates

  • Persistent foci (>24 hr): Unrepaired or complex damage

  • Rapid resolution: Efficient repair, simple breaks

• Contextual data integration:

  • Combine with cell cycle analysis

  • Consider p53 status of cells

  • Integrate with other DNA damage markers

  • Correlate with cellular outcomes (survival, senescence)