Phospho-Histone H2AX (S139) Recombinant Monoclonal Antibody

What is γ-H2AX and why is it important in DNA damage research?

γ-H2AX refers to the histone variant H2AX when phosphorylated at serine 139, which occurs rapidly following double-stranded DNA breaks. This phosphorylation happens in response to various DNA-damaging agents including ionizing radiation, chemotherapeutic drugs, and endogenous physiological processes. The importance of γ-H2AX stems from its role in triggering cell cycle arrest and DNA damage response repair mechanisms, making it an invaluable biomarker for studying genome stability, cell cycle regulation, and DNA repair pathways .

When DNA double-strand breaks occur, H2AX becomes phosphorylated by kinases such as ATM, ATR, and DNA-PK. This modification serves as a signal to recruit DNA repair proteins to the damage site, forming visible nuclear foci that can be detected through various immunological methods. The ability to visualize and quantify these foci provides researchers with a sensitive tool for measuring DNA damage at the cellular level .

What detection methods are available for Phospho-Histone H2AX (S139) and how do they compare?

Multiple detection methods are available for γ-H2AX quantification, each with distinct advantages depending on your research questions:

Immunofluorescence (IF): Enables visualization of discrete γ-H2AX foci within individual nuclei. This method provides spatial information about damage distribution and is highly sensitive for detecting even low levels of DNA damage. The technique typically involves fixing cells, permeabilization, antibody incubation, and counterstaining with DAPI for nuclear visualization .

Western Blot: Allows for semi-quantitative assessment of total γ-H2AX levels in cell populations. While less sensitive than IF (by approximately 16-fold according to comparative studies), Western blotting provides information about the total cellular response rather than individual foci .

HTRF (Homogeneous Time-Resolved Fluorescence): This plate-based method offers quantitative detection without requiring gels, electrophoresis, or protein transfer steps. HTRF uses two labeled antibodies—one specific for the phosphorylated motif and another that recognizes H2AX independent of its phosphorylation state. When both antibodies bind, they generate a FRET signal proportional to phosphorylated protein concentration .

Comparative analysis has demonstrated that HTRF cellular assays are at least 16-fold more sensitive than Western Blot for detecting γ-H2AX, making them particularly valuable for high-throughput screening applications or when sample material is limited .

How do I optimize immunofluorescence staining for γ-H2AX foci detection?

Successful immunofluorescence detection of γ-H2AX requires careful optimization of multiple parameters:

Fixation protocol: Use methanol-free formaldehyde (1% in PBS) at 0°C for 15 minutes, followed by transfer to 70% ethanol for at least 2 hours at -20°C. This preserves nuclear architecture while maintaining antibody epitope accessibility .

Permeabilization: Treat with 0.1% Triton X-100 in PBS for 5 minutes on ice to ensure antibody penetration without disrupting nuclear morphology .

Blocking: Apply 1% BSA in PBS for 30 minutes to reduce non-specific binding .

Antibody concentration: For primary antibodies, optimal dilutions typically range from 1:800 to 1:2500 depending on the specific antibody and application. For the Human Phospho-Histone H2AX (S139) Antibody, concentrations of 5-10 μg/mL have been validated for fluorescence immunocytochemistry .

Incubation conditions: Primary antibody incubation should be performed for 2-3 hours at room temperature or overnight at 4°C .

Secondary antibody selection: Fluorescent-conjugated secondary antibodies such as NorthernLights™ 557-conjugated Anti-Rabbit IgG provide strong signal with low background. Counterstain with DAPI to visualize nuclei .

For adherent cells, follow standard coverslip staining protocols. For suspension cells, use cytocentrifugation to attach cells to slides before proceeding with the staining protocol .

What controls should I include when measuring γ-H2AX in experimental settings?

Proper controls are essential for reliable γ-H2AX analysis:

Positive controls: Include samples treated with known DNA-damaging agents such as:

• Etoposide (25 μM for 4 hours)

• Camptothecin (1 μM for 24 hours)

• Neocarzinostatin (various concentrations for 30 minutes)

• Ultraviolet radiation

Negative controls: Untreated cells processed identically to experimental samples are crucial for establishing baseline phosphorylation levels .

Antibody controls: Include samples without primary antibody to assess secondary antibody non-specific binding.

Cell cycle normalization: Since DNA and histone content vary through the cell cycle, normalize γ-H2AX measurements by cell cycle phase. For microscopy-based quantification, multiplying S-phase values by 0.75 and G2/M values by 0.5 can compensate for increased DNA/histone content during cycle progression .

How can I distinguish between DNA damage-induced (DI) and apoptosis-associated (AA) H2AX phosphorylation?

Distinguishing between DNA damage-induced (DI) and apoptosis-associated (AA) H2AX phosphorylation is methodologically challenging but crucial for accurate data interpretation. These two types of phosphorylation represent distinct cellular processes:

Temporal analysis: DI H2AX phosphorylation typically occurs rapidly (within minutes to hours) after DNA damage, while AA phosphorylation appears later during apoptotic execution (typically 24+ hours after damage). Time-course experiments can help distinguish these responses .

Pattern analysis: DI phosphorylation typically appears as discrete nuclear foci corresponding to individual break sites, while AA phosphorylation often presents as pan-nuclear staining or peripheral nuclear staining with more intense, homogeneous signals .

Co-staining approaches: Combine γ-H2AX staining with markers of apoptosis (such as cleaved caspase-3 or TUNEL) to differentiate cells undergoing programmed cell death from those experiencing DNA damage repair .

Intensity thresholding: The mean IF intensity of DI H2AX phosphorylation is generally lower than that of AA phosphorylation. Setting appropriate thresholds during image analysis can help distinguish these populations .

Cell morphology: Apoptotic cells typically show characteristic morphological changes including nuclear condensation and fragmentation, which can be observed via DAPI staining. These features can help identify cells with AA phosphorylation .

What is the temporal relationship between DNA damage and γ-H2AX foci formation, and how should this influence experimental design?

Understanding the temporal dynamics of γ-H2AX is critical for experimental planning:

Initial phosphorylation: γ-H2AX foci can be detected as early as 1 hour after DNA damage induction, making it a rapid response biomarker .

Peak response: For many DNA-damaging agents, peak γ-H2AX levels occur approximately 24 hours after exposure, even though the actual DNA damage (such as crosslinking) may peak earlier. This is particularly evident with crosslinking agents like SJG-136, where maximum γ-H2AX response was observed 24 hours after the peak of crosslinking .

Persistence: Unlike many other DNA damage markers, γ-H2AX can persist for extended periods. In clinical studies with the DNA crosslinking agent SJG-136, significant levels of foci were still evident at days 8 and 15 post-treatment, consistent with the persistence of the DNA damage .

Cumulative effects: With repeated treatments, γ-H2AX responses can be cumulative. In clinical trials with a daily × 3 schedule of SJG-136, foci formation was observed before the third dose in cycle 1, and an increased response was evident during the second cycle .

These temporal considerations suggest the following experimental design principles:

• Include multiple timepoints (1h, 4h, 24h, and later if possible) to capture the full dynamics of the response

• For crosslinking agents, expect delayed peak responses compared to direct DNA-breaking agents

• For longitudinal or repeated treatment studies, account for residual γ-H2AX from previous exposures

• Consider tissue-specific differences in response timing (tumor biopsies showed higher levels than lymphocytes at 4 hours post-infusion in clinical studies)

How can γ-H2AX be effectively used as a pharmacodynamic marker in clinical trials?

γ-H2AX has proven valuable as a pharmacodynamic marker in clinical trials, particularly for DNA-damaging therapeutics. Implementation requires careful consideration of several factors:

Sensitivity advantage: γ-H2AX foci measurement has been demonstrated to be more than 10-fold more sensitive than traditional methods like the Comet assay for detecting DNA damage, making it particularly valuable for monitoring low-dose drug effects .

Tissue selection: While peripheral blood lymphocytes provide an easily accessible surrogate tissue, tumor biopsies show higher levels of γ-H2AX induction (as observed in clinical studies where tumor samples showed greater foci formation than lymphocytes from the same patients) .

Standardized protocols: Implement rigorous standardization for sample collection, processing time, fixation methods, and quantification approaches to ensure comparable results across multiple timepoints and patients .

Quantification methodology:

• For foci counting, automated image analysis systems can reduce observer bias

• Report both percentage of positive cells and average foci number per cell

• For flow cytometry approaches, report mean fluorescence intensity normalized to control samples

Correlation with outcomes: Connect γ-H2AX measurements with clinical responses, documenting both immediate pharmacodynamic effects and long-term patient outcomes .

Limitations to consider:

• The relationship between γ-H2AX levels and administered drug dose is not always linear

• Inter-patient variability in baseline and induced γ-H2AX levels can be substantial

• The timing of peak γ-H2AX response may vary depending on the mechanism of action of the therapeutic agent

A clinical trial example examining γ-H2AX as a pharmacodynamic marker for the DNA crosslinking agent SJG-136 demonstrated that foci were detectable in lymphocytes 1 hour post-administration, with maximum response at 24 hours, and persistence through days 8 and 15, consistent with the known persistence of DNA damage from this agent .

How do different cell-based detection platforms for γ-H2AX compare in terms of sensitivity and throughput?

Various platforms offer different advantages for γ-H2AX detection:

HTRF assay vs. Western Blot:
The HTRF (Homogeneous Time-Resolved Fluorescence) assay demonstrates significant advantages over Western Blot for high-throughput applications. Comparative analysis using serial dilutions of HEK-293 cell lysates showed that HTRF cellular assays were at least 16-fold more sensitive than Western Blot for detecting phosphorylated H2AX (Ser139) .

Plate-based protocols:
HTRF offers two distinct protocols:

• 2-plate protocol: Cells are cultured in a 96-well plate, lysed, and transferred to a 384-well low volume detection plate before adding detection reagents. This approach allows monitoring of cell viability and confluence .

• Single-plate protocol: Culturing, stimulation, and lysis occur in a single plate with no washing steps required. This HTS-designed protocol enables miniaturization while maintaining robust signal quality .

Dose-response sensitivity:
In validation studies using Jurkat cells treated with increasing concentrations of neocarzinostatin (30 min exposure), HTRF detection showed clear dose-dependent increases in γ-H2AX signal after overnight incubation with detection reagents .

The following table summarizes key differences between detection methods:

For researchers requiring high-throughput capability with maximal sensitivity, HTRF offers significant advantages, while immunofluorescence remains essential for spatial analysis of damage patterns within individual cells .