Anti-Phospho-Histone H2A.X (Ser139) (2A9) Mouse Monoclonal Antibody: Application and Best Practices

What This Product Solves

The Anti-Phospho-Histone H2A.X (Ser139) (2A9) Mouse Monoclonal Antibody (SKU: MA4415) addresses the need for reliable, phospho-specific detection of histone H2A.X phosphorylated at serine 139 (gamma-H2A.X). This modification is a widely recognized marker for DNA double-strand breaks, pivotal in DNA damage response and repair studies. The antibody is designed for use in Western blotting (WB) and immunocytochemistry/immunofluorescence (ICC/IF), providing a targeted solution for researchers investigating DNA repair mechanisms, chromatin remodeling, and related signaling pathways in human and mouse samples. The product is not suitable for detecting non-phosphorylated H2A.X or applications outside the specified species and techniques.

Protocol Parameters

Protocol Parameters

• Assay: Western blot (WB) | Value: 0.5–2 μg/mL | Applicability: Human or mouse cell/tissue lysates | Rationale: Recommended starting range for detecting gamma-H2A.X with minimal background based on standard workflow optimization | Source_type: workflow_recommendation
• Assay: Immunocytochemistry/immunofluorescence (ICC/IF) | Value: 1–5 μg/mL | Applicability: Fixed human or mouse cells | Rationale: Empirical working range for ICC/IF to ensure specific nuclear staining while minimizing non-specific signal | Source_type: workflow_recommendation
• Storage temperature | Value: –20°C | Applicability: All antibody aliquots | Rationale: Maintains antibody stability and preserves function for 12 months; avoid repeated freeze-thaw cycles | Source_type: product_spec | Source_link: https://www.apexbt.com/anti-phospho-histone-h2a-x-ser139-2a9-mouse-monoclonal-antibody.html
• Buffer composition | Value: PBS, 50% glycerol, 0.5% BSA, 0.02% sodium azide, pH 7.3 | Applicability: Supplied antibody formulation | Rationale: Stabilizes antibody and reduces freeze-thaw damage | Source_type: product_spec | Source_link: https://www.apexbt.com/anti-phospho-histone-h2a-x-ser139-2a9-mouse-monoclonal-antibody.html

Workflow Setup and QC Checklist

• Sample Preparation: Use freshly prepared or appropriately stored lysates/fixed cells from human or mouse sources. Confirm that samples have undergone treatments (e.g., irradiation, genotoxic stress) expected to induce DNA double-strand breaks if positive signal is required.
• Antibody Handling: Thaw antibody aliquots on ice. Mix gently but thoroughly before use. Avoid repeated freeze-thaw cycles; aliquot upon first thaw if multiple uses are expected.
• Controls: Include untreated/negative controls (no DNA damage) and, when possible, total H2A.X or loading controls for Western blot to distinguish phospho-specific from general histone signal.
• Incubation: For WB, incubate primary antibody overnight at 4°C with gentle agitation. For ICC/IF, incubate for 1–2 h at room temperature or overnight at 4°C, depending on protocol stringency needs.
• Detection: Use species-appropriate, high-sensitivity secondary antibodies; verify compatibility with unconjugated mouse IgG2a primary antibody. Optimize washing steps to minimize background.
• Documentation: Record antibody lot number, dilution, incubation times, and sample treatment details for reproducibility.

Common Failure Modes and Fixes

• High background in WB or ICC/IF: Confirm specificity of secondary antibodies, increase washing stringency, or lower primary antibody concentration within recommended range (see Protocol Parameters).
• Weak or absent gamma-H2A.X signal: Ensure DNA damage induction in positive controls, verify sample integrity, and increase antibody concentration incrementally (do not exceed recommended ICC/IF range).
• Non-specific nuclear staining: Apply additional blocking (e.g., 5% BSA or serum), and confirm that fixation/permeabilization protocols do not compromise epitope accessibility.
• Loss of activity after storage: Adhere strictly to –20°C storage and avoid freeze-thaw cycles. Use aliquots to reduce degradation risk.

Scope and Limitations

• This mouse monoclonal antibody against phospho-H2A.X is validated for use in human and mouse samples only. Efficacy on other species or in vivo imaging has not been established by product specification.
• The antibody is not intended for detection of total H2A.X or other histone modifications, nor is it conjugated for direct detection; appropriate secondary antibodies are required.
• Phospho-specific recognition is contingent on proper sample treatment and preservation of the phospho-epitope; harsh fixation or prolonged storage in suboptimal conditions may abrogate signal.
• No direct peer-reviewed evidence is available for SKU MA4415; application parameters are based on product dossier and general workflow best practices. Researchers requiring cross-reactivity data or validation in non-canonical assays should perform independent pilot testing.

Conclusion

The Anti-Phospho-Histone H2A.X (Ser139) (2A9) Mouse Monoclonal Antibody from APExBIO is a practical tool for detecting gamma-H2A.X, supporting quantitative and qualitative analyses of DNA double-strand break responses in human and mouse cells by WB and ICC/IF. Adhering to protocol parameters and QC measures enables reproducible, phospho-specific results. For further technical details, refer to the product page or contact APExBIO technical support.