Phospho-Histone H2A (Ser129) Antibody
Description
Fundamentals of Phospho-Histone H2A (Ser129) Antibody
Phospho-Histone H2A (Ser129) Antibody is a specialized immunological reagent designed to specifically recognize and bind to histone H2A proteins that have been phosphorylated at the serine 129 position. This particular post-translational modification plays a crucial role in DNA double-strand break (DSB) repair mechanisms. The antibody serves as an essential research tool for investigating DNA damage signaling pathways, particularly those involved in genomic stability maintenance. These antibodies are available in various formats, including unconjugated forms and fluorescent conjugates such as FITC and PerCP-Cy5.5, enabling their application across diverse experimental techniques .
Histone H2A constitutes one of the core components of nucleosomes, which are fundamental units of chromatin structure. Nucleosomes function to wrap and compact DNA, thereby regulating its accessibility to cellular machinery involved in essential processes like transcription, replication, and repair. The phosphorylation of H2A at serine 129 represents a critical cellular response to DNA damage, particularly double-strand breaks, making antibodies against this modification invaluable tools for studying genomic integrity mechanisms .
The phosphorylation of histone H2A at serine 129 represents one of the earliest cellular responses to DNA double-strand breaks. This modification, often referred to as γ-H2A(X) in the literature, plays a fundamental role in the DNA damage response pathway.
Role in DNA Double-Strand Break Repair
Research in yeast models has demonstrated that H2A Ser129 is essential for the efficient repair of DNA double-stranded breaks (DSBs) during replication. Following DNA damage, this residue becomes rapidly phosphorylated, forming distinct nuclear foci that serve as recruitment platforms for DNA repair factors .
The phosphorylation event is catalyzed by PI(3)K-like kinases, specifically Mec1 and Tel1 in yeast (orthologous to mammalian ATR and ATM, respectively). These kinases display partial redundancy in their function, as studies have shown that while the presence of either kinase permits substantial γ-H2A(X) formation, strains lacking both Mec1 and Tel1 are unable to generate this modification .
Comparative Analysis Across Species
The importance of H2A serine phosphorylation in DNA damage response is evolutionarily conserved, though with interesting species-specific variations:
| Species | DNA content (Mbp) | γ-H2A(X)/total H2A(X) (%) | DSBs/Gy | γ-H2A(X)/DSB (%) | Focus size (Mbp) | Molecules of γ-H2A(X) per DSB |
|---|---|---|---|---|---|---|
| Yeast (S. cerevisiae) | 17 | 4 | 0.1 | 0.02 | 0.004 | 34 |
| Human (H. sapiens) | 6,600 | 25 | 33 | 0.03 | 2 | 1,800 |
Research Applications in DNA Damage Response Studies
Phospho-Histone H2A (Ser129) antibodies have proven invaluable for investigating the molecular mechanisms of DNA damage response and repair pathways.
Western Blot Applications
The primary application for these antibodies is Western blotting, where they enable quantitative assessment of H2A Ser129 phosphorylation levels in response to various genotoxic agents or genetic mutations. Typically used at dilutions ranging from 1:300 to 1:5000, these antibodies provide sensitive detection of phosphorylated H2A, allowing researchers to monitor DNA damage signaling dynamics .
Key Research Findings Using Phospho-H2A (Ser129) Antibodies
Studies utilizing these antibodies have yielded several significant discoveries regarding the functional role of H2A Ser129 phosphorylation:
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Checkpoint Independence: Research has demonstrated that H2A Ser129 phosphorylation functions independently of the RAD9 and RAD24 checkpoint pathways. This was evidenced by the lack of epistasis between h2a-s129a mutations and rad24Δ or rad9Δ checkpoint-deficient strains, indicating that γ-H2A(X) contributes to DNA repair through mechanisms distinct from canonical checkpoint activation .
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Repair Pathway Integration: Genetic studies revealed that H2A Ser129 functions within the same pathways as Rad52 and Mre11, key components of homologous recombination repair. When h2a-s129a mutations were introduced into rad52Δ or mre11Δ repair-deficient strains, no additive sensitivity to DNA-damaging agents was observed, suggesting functional integration within these repair pathways .
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"On-the-fly" Repair Mechanism: Perhaps most significantly, H2A Ser129 phosphorylation has been identified as essential for efficient "on-the-fly" repair of DNA damage during ongoing replication. This function is particularly crucial for addressing lesions that do not activate the intra-S-phase checkpoint, highlighting a unique role for this histone modification in maintaining genomic integrity during DNA synthesis .
Immunogen Design and Antibody Production
The specificity of these antibodies stems from careful immunogen design. For instance, BIOSS products utilize "KLH conjugated synthetic peptide derived from yeast Histone H2A around the phosphorylation site of S129" as the immunogen, ensuring targeted recognition of the phosphorylated serine residue .
The production process typically involves immunizing rabbits with these synthetic phosphopeptides, followed by affinity purification to isolate antibodies with high specificity for the phosphorylated epitope. Monoclonal versions undergo additional cloning and selection procedures to identify single B-cell clones producing highly specific antibodies .
Molecular Mechanisms and Pathway Integration
The phosphorylation of histone H2A at serine 129 initiates a complex cascade of molecular events that facilitate efficient DNA repair.
Kinase-Mediated Phosphorylation
In response to DNA double-strand breaks, PI(3)K-like kinases (Mec1/Tel1 in yeast, ATM/ATR/DNA-PK in mammals) rapidly phosphorylate H2A at serine 129 within the conserved C-terminal motif Ala-Ser-Gln-Glu-Leu. This phosphorylation occurs preferentially on nucleosomes adjacent to break sites, creating a specialized chromatin environment that facilitates repair factor recruitment .
Integration with DNA Repair Pathways
Genetic studies utilizing h2a-s129a mutants (where serine 129 is replaced with alanine, preventing phosphorylation) have provided valuable insights into how this modification interfaces with established DNA repair pathways:
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Homologous Recombination: The h2a-s129a mutation does not enhance sensitivity to DNA-damaging agents in rad52Δ or mre11Δ backgrounds, indicating epistasis and suggesting that H2A Ser129 phosphorylation functions within the homologous recombination repair pathway .
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Checkpoint Pathways: Conversely, h2a-s129a mutations substantially decrease survival in rad24Δ or rad9Δ checkpoint-deficient backgrounds, with even greater sensitivity observed in double mutants. This lack of epistasis demonstrates that H2A Ser129 phosphorylation operates independently of these checkpoint mechanisms, potentially providing an alternative repair strategy when checkpoint functions are compromised .
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Topoisomerase I-Mediated Damage: Research with camptothecin (CPT), which stabilizes topoisomerase I-DNA complexes, revealed that h2a-s129a mutants exhibit increased sensitivity to this agent. This sensitivity requires the active S-enantiomer of CPT and the presence of TOP1, confirming a specific role for H2A Ser129 phosphorylation in addressing topoisomerase-induced DNA damage .
Product Specs
Q&A
What is Phospho-Histone H2A (Ser129) and why is it significant in research?
Histone H2A phosphorylated at Serine 129 (Ser129) is a key biomarker in DNA damage response pathways, particularly in response to double-strand breaks (DSBs). This post-translational modification plays a crucial role in recruiting DNA repair machinery to damage sites. In yeast, H2A Ser129 phosphorylation is equivalent to the mammalian H2AX Ser139 phosphorylation (γ-H2AX), making it an essential target for studying DNA damage response mechanisms across species .
What forms of Phospho-Histone H2A (Ser129) antibodies are commercially available?
These antibodies are available in several formats:
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Polyclonal antibodies from rabbit hosts, purified by affinity chromatography
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Monoclonal antibodies, including recombinant rabbit monoclonal formats
What is the typical composition of commercial Phospho-Histone H2A (Ser129) antibody preparations?
Most preparations have the following characteristics:
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Buffer composition: PBS with 0.02% sodium azide as preservative and 50% glycerol, pH 7.4
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Purification method: Affinity chromatography using epitope-specific phosphopeptides
Technical Applications and Methodologies
For optimal visualization of DNA damage foci:
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Fix cells with 1% formaldehyde after DNA damage induction
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Wash cells thoroughly and treat with zymolyase (10 μg/ml)
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Spot treated cells onto poly-l-lysine-coated slides
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Incubate with anti-phospho-histone H2A (Ser129) primary antibody (1:1,000 dilution)
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Wash with PBS and apply appropriate fluorophore-conjugated secondary antibody (e.g., Alexa Fluor 488-conjugated, 1:500 dilution)
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Counterstain nuclei with propidium iodide (2 μg/ml) after RNase A treatment
What positive controls should be used to validate antibody specificity?
The most effective controls include:
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MMS (methyl methanesulfonate) treated yeast nuclear extracts show increased signal compared to untreated extracts
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DNA damage-inducing agents like galactose (in appropriate genetic backgrounds)
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Peptide competition assays comparing phosphorylated vs. non-phosphorylated peptides
Species Reactivity and Cross-Reactivity
The phosphorylation site at Ser129 and surrounding amino acids are highly conserved between different species, particularly in the epitope sequence region. This conservation allows certain antibodies to recognize the phosphorylated form across species. The typical epitope sequence surrounding the phosphorylation site is K-A-Q-S(p)-K, which is similar in yeast and mammalian systems .
How should samples be prepared for optimal detection of Phospho-Histone H2A (Ser129)?
For optimal signal:
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Include phosphatase inhibitors in all extraction buffers to prevent dephosphorylation
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For nuclear extracts: separate nuclei prior to lysis to concentrate the histone fraction
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For Western blotting: acid extraction of histones can improve signal strength
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For immunofluorescence: gentle fixation (1% formaldehyde) preserves epitope accessibility
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For yeast studies: enzymatic digestion with zymolyase (10 μg/ml) facilitates antibody access
What is the optimal timing for detecting Phospho-Histone H2A (Ser129) after DNA damage induction?
Phosphorylation of H2A at Ser129 typically occurs rapidly after DNA damage:
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Initial phosphorylation is detectable within 15-30 minutes of damage induction
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Peak signal is generally observed between 1-4 hours post-damage
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In yeast, galactose-induced damage shows significant γ-H2A foci formation after 1 hour of induction
Timing should be optimized based on the specific damage induction method and cell type being studied.
What are the recommended storage conditions for Phospho-Histone H2A (Ser129) antibodies?
For maximum stability and activity:
How can antibody aliquoting be optimized to maintain long-term stability?
To maintain stability:
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Upon receipt, divide the antibody into small single-use aliquots
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Use sterile microcentrifuge tubes
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Quick-freeze aliquots in liquid nitrogen before storing at -20°C or -80°C
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Avoid more than 2-3 freeze-thaw cycles
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When thawing, keep on ice and return unused portions to -20°C immediately
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Include carrier proteins (BSA, 0.5-1%) if diluting before aliquoting
How can Phospho-Histone H2A (Ser129) antibody be used to map genomic sites of DNA damage?
For genome-wide mapping of DNA damage:
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Chromatin immunoprecipitation (ChIP) with the antibody can be performed to isolate DNA fragments associated with phosphorylated H2A
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The precipitated DNA can be analyzed by sequencing (ChIP-seq) to identify genomic regions with damage
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For yeast studies, yeast-specific protocols using zymolyase treatment before cell lysis are recommended
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A 1:100 dilution has been demonstrated effective for immunoprecipitation from yeast extracts
How does the detection of Phospho-Histone H2A (Ser129) compare between different types of DNA damage?
Different DNA damage induction methods can result in varying patterns:
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Double-strand breaks (DSBs) from ionizing radiation or radiomimetic drugs produce distinct foci
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Replication stress from hydroxyurea or aphidicolin results in more diffuse staining
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DNA crosslinking agents may produce unique patterns
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MMS treatment in yeast has been demonstrated to significantly increase phosphorylation at Ser129
The antibody can detect differences in these patterns, making it valuable for distinguishing damage mechanisms.
What are common causes of weak or no signal when using Phospho-Histone H2A (Ser129) antibody?
Common issues and solutions:
How can background or non-specific staining be reduced when using the antibody?
To reduce background:
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Increase blocking time and protein concentration (5% BSA or milk)
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Include 0.1-0.3% Triton X-100 in blocking and antibody dilution buffers
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Use longer washing steps and more washing buffer
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Optimize primary antibody concentration through titration experiments
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Pre-absorb antibody with non-target proteins if cross-reactivity is suspected
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For tissues, include an additional blocking step with normal serum from the same species as the secondary antibody
How is Phospho-Histone H2A (Ser129) related to DNA repair mechanisms?
Phosphorylation of H2A at Ser129 serves multiple functions in DNA repair:
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Acts as an early marker of DNA double-strand breaks
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Recruits repair proteins to damage sites
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Helps maintain repair factors at damage sites
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Facilitates chromatin remodeling necessary for repair
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In yeast, it recruits repair proteins similarly to how γ-H2AX functions in mammals
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Participates in the spindle checkpoint response to DNA damage
What is the relationship between Phospho-Histone H2A (Ser129) in yeast and γ-H2AX (Ser139) in mammals?
These modifications are functional homologs:
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Both are rapidly phosphorylated at serine residues in response to DNA damage
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Both serve as platforms for recruiting DNA repair factors
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Both can extend for kilobases from the actual break site
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Both are involved in checkpoint activation
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Phospho-H2A (Ser129) in yeast is performed by Mec1 and Tel1 kinases
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γ-H2AX (Ser139) in mammals is phosphorylated by ATM, ATR, and DNA-PK kinases
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The antibodies raised against either modification are generally specific to their respective targets and do not cross-react
