H2AFX Recombinant Monoclonal Antibody

What is H2AFX and why is it important in cellular biology?

H2AFX (also known as Histone H2AX or H2A.X) is a member of the histone H2A family, one of the four core histones involved in nucleosome formation and chromatin compaction. The critical importance of H2AFX lies in its role in DNA damage response pathways. When phosphorylated at serine 139 (creating γ-H2AX), it serves as a key marker of DNA double-strand breaks, enabling the recruitment of DNA repair proteins to damaged sites and contributing significantly to genomic integrity maintenance and cell viability . This phosphorylation occurs rapidly after DNA damage and creates a platform for the assembly of DNA repair complexes, making it an essential component in cellular mechanisms that protect against genomic instability.

How are H2AFX recombinant monoclonal antibodies produced?

H2AFX recombinant monoclonal antibodies are produced through a sophisticated process involving recombinant DNA technology and in vitro cloning. The process begins with cloning H2AFX antibody genes into expression vectors. These vectors are then introduced into host cells, creating a controlled environment for the recombinant antibody's expression within a cell culture system. Following expression, the antibody undergoes purification via affinity chromatography to ensure high specificity and consistency . Some H2AFX antibodies are generated using synthesized peptides derived from human H2AFX as the immunogen, resulting in highly specific non-conjugated rabbit IgG antibodies that are affinity-chromatography purified . This recombinant approach offers significant advantages over traditional monoclonal antibody production, including greater batch-to-batch consistency and reduced use of animals in production.

What are the primary applications of H2AFX recombinant monoclonal antibodies in research?

H2AFX recombinant monoclonal antibodies have diverse research applications, primarily in detecting human histone H2AX and its phosphorylated form. The antibodies are validated for multiple techniques:

• ELISA (Enzyme-Linked Immunosorbent Assay): For quantitative detection of H2AFX in solution

• IHC (Immunohistochemistry): For visualizing H2AFX in tissue sections, typically at dilutions of 1:50-1:200

• FC (Flow Cytometry): For analyzing H2AFX expression in cell populations, also at recommended dilutions of 1:50-1:200

• Western Blot: For detecting H2AFX in cell lysates and studying its modification states

• Immunoprecipitation (IP): For isolating H2AFX and associated protein complexes

• Immunofluorescence (IF): For subcellular localization studies and visualization of γ-H2AX foci formation

These applications enable researchers to investigate DNA damage response pathways, genotoxicity, cancer biology, and cellular responses to radiation and chemotherapeutic agents.

How do different phosphorylation sites on H2AFX affect antibody selection for specific research questions?

While phosphorylation at serine 139 (S139) of H2AFX is the most extensively studied modification, creating the γ-H2AX marker, researchers should consider that H2AFX contains multiple potential phosphorylation sites that may influence experimental outcomes and antibody selection. The phosphorylation at S139 (sometimes numbered as S140 depending on the reference sequence, as noted in entry NP_002096.1, Gene ID 3014 ) is critically important for DNA damage signaling.

When selecting an antibody for research:

• For DNA damage studies: Antibodies specifically recognizing phospho-S139 (γ-H2AX), such as the recombinant monoclonal rabbit antibodies described in the search results, are optimal .

• For total H2AFX studies: Non-phospho-specific antibodies that detect H2AFX regardless of modification state should be used.

• For multi-modification studies: Consider that other modifications (e.g., ubiquitination, acetylation) may occur simultaneously with phosphorylation and potentially affect antibody binding.

Advanced research may require antibodies that can distinguish between different combinations of post-translational modifications on H2AFX, necessitating careful validation in the specific experimental system being studied.

What are the key considerations when using H2AFX antibodies for quantifying DNA damage across different cell types?

Quantifying DNA damage using H2AFX antibodies presents several important considerations that vary across cell types:

Baseline Expression Variability:
Different cell types exhibit varying baseline levels of H2AFX expression, which must be normalized appropriately when comparing DNA damage responses. Stem cells and rapidly dividing cancer cells typically show higher baseline H2AFX levels than terminally differentiated cells.

Kinetics of γ-H2AX Formation and Resolution:
The temporal dynamics of γ-H2AX formation and resolution after DNA damage differ significantly between cell types:

• Lymphocytes: Typically show rapid γ-H2AX formation (minutes) and clearance (hours)

• Neurons: Often exhibit prolonged γ-H2AX retention (days)

• Cancer cells: May show aberrant γ-H2AX kinetics due to defective DNA repair pathways

Methodological Standardization:
For accurate quantification:

• Use consistent fixation protocols (paraformaldehyde concentration and duration)

• Standardize antibody dilutions (1:50-1:200 range is commonly effective for IHC and FC applications)

• Implement appropriate permeabilization methods specific to the cell type

• Include positive controls (e.g., irradiated cells) and negative controls

Data Interpretation:
When analyzing results, consider that γ-H2AX signal intensity may not linearly correlate with the number of DNA breaks across all cell types due to differences in chromatin accessibility and H2AFX density in the genome.

How can H2AFX antibodies be used to distinguish between different types of DNA damage responses?

H2AFX antibodies can be powerful tools for distinguishing between different types of DNA damage responses when used in sophisticated experimental designs:

Temporal Pattern Analysis:

• Replication stress: Typically produces focal γ-H2AX patterns during S-phase

• Double-strand breaks (DSBs): Generate distinct γ-H2AX foci at break sites

• Apoptosis: Creates pan-nuclear γ-H2AX staining pattern

Co-localization Studies:
Combining γ-H2AX detection with other DNA damage response proteins reveals specific pathway activation:

• 53BP1 + γ-H2AX: Non-homologous end joining (NHEJ) repair

• RAD51 + γ-H2AX: Homologous recombination (HR) repair

• BRCA1 + γ-H2AX: HR pathway activation

• RPA + γ-H2AX: Replication stress and single-strand DNA exposure

Pulse-Chase Experiments:
Using time-course analysis with γ-H2AX antibodies can distinguish between:

• Acute, high-dose damage (rapid formation and resolution)

• Chronic, low-dose exposure (persistent low-level signal)

• Defective repair (abnormal persistence of foci)

Multi-parameter Analysis:
Combining phospho-specific H2AFX antibodies with cell cycle markers (e.g., PCNA, Ki67) and other DNA damage markers enables comprehensive characterization of the damage response type within specific cell populations and cell cycle phases.

What are the optimal protocols for using H2AFX recombinant monoclonal antibodies in different applications?

Western Blot Protocol Optimization:

• Sample preparation: Use 5% Milk-TBST for blocking and as antibody diluent

• Primary antibody incubation: Overnight at 4°C

• Secondary antibody selection: For cell lysates, use Goat anti-Rabbit IgG Heavy and Light Chain Antibody

• For immunoprecipitates: Use Goat anti-Rabbit Light Chain HRP Conjugate with 5% Normal Pig Serum added to the blocking buffer

Immunohistochemistry (IHC) Optimization:

• Recommended dilution range: 1:50-1:200

• Antigen retrieval method: Heat-induced epitope retrieval in citrate buffer pH 6.0

• Detection system: Polymer-based HRP detection systems are preferred for signal amplification

• Counterstaining: Light hematoxylin to visualize nuclear localization

Flow Cytometry (FC) Protocol:

• Cell fixation: 2-4% paraformaldehyde for 10-15 minutes

• Permeabilization: 0.1-0.5% Triton X-100 or 70% ethanol (for improved nuclear access)

• Antibody dilution: 1:50-1:200

• Incubation time: 1 hour at room temperature or overnight at 4°C

• Washing: PBS with 0.1% Tween-20, three times between steps

ELISA Protocol Considerations:

• Coating concentration: 1-5 μg/ml of capture antibody

• Blocking: 1-3% BSA in PBS

• Sample preparation: Prepare cell or tissue lysates with appropriate extraction buffer

• Detection: Use appropriate HRP-conjugated secondary antibody and substrate

What controls should be included when performing experiments with H2AFX antibodies?

Positive Controls:

• For γ-H2AX antibodies: Include cells treated with ionizing radiation (2-10 Gy) or radiomimetic drugs (e.g., bleomycin, etoposide)

• For total H2AFX antibodies: Use cell lines with confirmed H2AFX expression (most human cell lines express detectable levels)

• Consider including known responsive cell lines such as lymphoblasts or fibroblasts

Negative Controls:

• Primary antibody omission: Replace primary antibody with isotype-matched IgG (Rabbit IgG for most H2AFX recombinant monoclonal antibodies)

• Blocking peptide: Pre-incubate antibody with the immunizing peptide to demonstrate specificity

• H2AFX knockout/knockdown cells (if available): Demonstrate antibody specificity

Technical Controls:

• Dilution series: Test multiple antibody dilutions (1:50, 1:100, 1:200) to determine optimal signal-to-noise ratio

• Incubation time variants: Compare results with different incubation times

• Storage impact: Test antibody performance after different storage conditions

Biological Response Controls:

• Dose response: Include samples treated with different doses of DNA damaging agents

• Time course: Collect samples at various time points after treatment to capture dynamics

• Pharmacological inhibitors: Include ATM/ATR kinase inhibitors to block H2AFX phosphorylation

How can researchers optimize H2AFX antibody dilutions and incubation conditions for maximum sensitivity and specificity?

Optimizing H2AFX antibody protocols requires systematic testing of multiple parameters to achieve the ideal balance between sensitivity and specificity:

Antibody Dilution Optimization:

• Start with the manufacturer's recommended range (typically 1:50-1:200 for IHC and FC)

• Prepare a dilution series (e.g., 1:25, 1:50, 1:100, 1:200, 1:400)

• Test each dilution on positive control samples

• Evaluate signal-to-noise ratio at each dilution

Incubation Time and Temperature Matrix:

Buffer Composition Variations:

• Test different blocking agents: BSA vs. normal serum vs. milk proteins

• Adjust detergent concentration (0.05% to 0.3% Tween-20) to balance membrane permeabilization and antibody retention

• Consider adding protein stabilizers (1-5% glycerol) for longer incubations

Antigen Retrieval Method Comparison:
For fixed tissues and cells, compare different antigen retrieval methods:

• Heat-induced epitope retrieval (HIER) in citrate buffer (pH 6.0)

• HIER in EDTA buffer (pH 8.0-9.0)

• Enzymatic retrieval with proteinase K

• No retrieval (for some applications)

Document results systematically to create a reference protocol optimized for your specific experimental system and application.

What are common problems encountered with H2AFX antibodies and how can they be resolved?

High Background Signal:

• Problem: Non-specific binding leading to diffuse background staining

• Solutions:

  • Increase blocking time (2-3 hours or overnight)

  • Use alternative blocking agents (switch between BSA, normal serum, milk)

  • Increase washing steps (duration and number)

  • Further dilute primary antibody

  • Ensure proper storage conditions to prevent antibody degradation (-20°C or -80°C)

Weak or No Signal:

• Problem: Insufficient antibody binding or detection

• Solutions:

  • Verify antigen is not degraded in samples

  • Optimize antigen retrieval for IHC/ICC

  • Use more concentrated antibody solution (within 1:50-1:200 range)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Try more sensitive detection systems (e.g., tyramide signal amplification)

  • Confirm sample handling preserves phosphorylation (add phosphatase inhibitors)

Variable Results Between Experiments:

• Problem: Inconsistent staining patterns or intensities

• Solutions:

  • Standardize all protocol steps (timing, temperature, reagent sources)

  • Prepare larger volumes of diluted antibody to use across experiments

  • Include internal reference samples in each experiment

  • Monitor and control fixation times precisely

  • Avoid repeated freeze-thaw cycles of antibody

Cross-Reactivity Issues:

• Problem: Antibody binds to unintended targets

• Solutions:

  • Validate antibody specificity using knockout/knockdown controls

  • Perform peptide competition assays

  • Try alternative recombinant monoclonal antibodies targeting different epitopes

  • Increase washing stringency (higher salt or detergent concentration)

How should researchers analyze and interpret γ-H2AFX foci formation data from different experimental conditions?

Analysis and interpretation of γ-H2AFX foci require systematic approaches to generate reliable and reproducible results:

Quantification Methods:

• Manual counting: Suitable for clear, distinct foci in limited samples

  • Count foci per nucleus (typically 30-100 nuclei per condition)

  • Establish consistent criteria for what constitutes a focus (size, intensity)

• Automated image analysis:

  • Use specialized software (ImageJ with FIJI plugins, CellProfiler, etc.)

  • Set consistent parameters (threshold, minimum size, intensity)

  • Validate automated counts against manual counts for subset of images

Statistical Approaches:

• For comparing treatments/conditions:

  • Use appropriate statistical tests based on data distribution

  • Consider both mean foci per nucleus and distribution patterns

  • Report both number of foci per cell and percentage of cells with foci

Temporal Analysis:

• γ-H2AFX foci kinetics reveal important information about DNA damage processing:

  • Initial response (30 minutes - 1 hour): Formation rate indicates damage recognition efficiency

  • Peak response (1-4 hours): Maximum foci represent total damage burden

  • Resolution phase (4-24 hours): Disappearance rate indicates repair capacity

Combined Parameter Analysis:
When integrating γ-H2AFX data with other parameters, consider:

• Cell cycle phase correlation (G1 vs. S vs. G2/M responses differ)

• Co-localization with other repair factors (53BP1, RAD51, etc.)

• Relationship between foci size/intensity and downstream outcomes (cell death, mutations)

Interpretation Guidelines:

• Small, numerous foci often indicate acute DNA damage (e.g., radiation)

• Large, persistent foci suggest complex or unrepairable damage

• Pan-nuclear staining typically indicates replication stress or pre-apoptotic state

• Foci number does not always linearly correlate with damage severity

What considerations are important when using H2AFX antibodies for multiplexed imaging with other DNA damage markers?

Multiplexed imaging combining H2AFX antibodies with other DNA damage markers provides rich contextual information but requires careful technical considerations:

Antibody Compatibility:

• Species origin conflicts: When using multiple primary antibodies, they must come from different host species or be directly conjugated to different fluorophores

  • The H2AFX recombinant monoclonal antibodies described are rabbit-derived , requiring companion antibodies from mouse, rat, or directly conjugated antibodies

• Cross-reactivity testing: Validate that secondary antibodies don't cross-react with inappropriate primaries

  • Test each antibody individually before combining them

Fluorophore Selection:

• Choose fluorophores with minimal spectral overlap

• Consider using Alexa Fluor 350-conjugated H2AFX antibodies as the blue channel component

• Reserve brighter fluorophores (Alexa 488, Cy3) for markers with lower expression

• Account for autofluorescence spectrum of your specific tissue/cell type

Sequential Staining Protocol:
When antibody conflicts cannot be avoided:

• Apply first primary antibody and corresponding secondary

• Fix the antibody-antigen complexes with 4% paraformaldehyde

• Block with excess unconjugated Fab fragments

• Apply subsequent primary-secondary pairs

• Include extensive washing between steps

Image Acquisition Settings:

• Capture single-stained controls to calculate and correct for spectral overlap

• Maintain consistent microscope settings across samples for comparative analysis

• Consider spectral unmixing for highly multiplexed imaging

Data Analysis Approaches:

• Co-localization analysis:

  • Calculate Pearson's or Mander's coefficients between γ-H2AFX and other repair factors

  • Use distance-based measurements to quantify spatial relationships between different foci

• Sequential recruitment analysis:

  • Design time-course experiments to determine ordering of factor recruitment

  • Analyze size-dependent co-localization to assess repair complex maturation

Controls for Multiplexed Imaging:

• Single antibody controls to assess bleed-through

• Fluorescence minus one (FMO) controls to set accurate thresholds

• Competing unlabeled antibodies to confirm specificity in the multiplexed context

Recommended Dilutions and Applications for H2AFX Recombinant Monoclonal Antibodies

Comparing γ-H2AFX Foci Formation in Response to Different DNA Damaging Agents

How might emerging technologies enhance the use of H2AFX antibodies in research?

H2AFX antibody technology continues to evolve with several promising developments that may enhance their research utility. Super-resolution microscopy techniques like STORM and PALM are now enabling visualization of individual γ-H2AFX foci at nanometer resolution, potentially revealing substructures within repair complexes that were previously undetectable. Mass cytometry (CyTOF) approaches using metal-tagged H2AFX antibodies allow simultaneous detection of dozens of other DNA damage response proteins in single cells, providing unprecedented comprehensive pathway analysis.

Live-cell imaging compatible H2AFX antibody fragments and nanobodies are being developed to track DNA damage responses in real-time without fixation artifacts. Furthermore, proximity ligation assays using H2AFX antibodies paired with antibodies against other repair factors can provide quantitative measures of protein interactions at damage sites. These technological advances will likely transform our understanding of DNA damage response dynamics and their relationship to disease processes and treatment responses.

What are the most significant recent discoveries involving H2AFX antibodies in cancer and aging research?

Recent research using H2AFX antibodies has yielded significant insights into cancer biology and aging processes. Studies have revealed that certain cancer types exhibit "γ-H2AFX signatures" that correlate with treatment resistance and poor prognosis, offering potential biomarkers for personalized therapy approaches. In aging research, persistent γ-H2AFX foci have been identified as markers of senescence-associated DNA damage in multiple tissues, supporting the DNA damage theory of aging.