Acetyl-HIST1H2AG (K36) Antibody

What is the Acetyl-HIST1H2AG (K36) Antibody and what epitope does it recognize?

The Acetyl-HIST1H2AG (K36) Antibody is a polyclonal antibody specifically designed to detect the acetylation of lysine 36 on histone H2A type 1 (HIST1H2AG). This antibody recognizes a peptide sequence surrounding the acetylated lysine 36 residue derived from human histone H2A type 1 . The specificity of this antibody for acetylated K36 makes it a valuable tool for studying post-translational modifications of histones, which play crucial roles in epigenetic regulation of gene expression. The antibody has been validated for multiple applications including ELISA, Western blotting, immunocytochemistry, immunofluorescence, and chromatin immunoprecipitation (ChIP) . Understanding the precise epitope recognition is essential for interpreting experimental results and designing appropriate controls when working with this antibody.

What are the validated applications for the Acetyl-HIST1H2AG (K36) Antibody?

The Acetyl-HIST1H2AG (K36) Polyclonal Antibody has been validated for multiple applications in molecular and cellular biology research. According to the product information, this antibody has demonstrated reliable performance in enzyme-linked immunosorbent assay (ELISA), Western blotting (WB), immunocytochemistry (ICC), immunofluorescence (IF), and chromatin immunoprecipitation (ChIP) . Each application requires specific optimization of experimental conditions including antibody dilution, incubation time, and buffer composition to achieve optimal signal-to-noise ratio. Western blotting can be used to detect and quantify the global levels of K36 acetylation on histone H2A, while ICC and IF allow visualization of the nuclear localization and distribution patterns of this modification. ChIP applications are particularly valuable for identifying genomic regions enriched with this specific histone modification, allowing researchers to associate H2A K36 acetylation with specific genes or regulatory elements .

What is the biological significance of acetylation at lysine 36 on histone H2A?

Acetylation at lysine 36 on histone H2A represents an important post-translational modification that contributes to the complex histone code regulating chromatin structure and gene expression. Histone acetylation generally neutralizes the positive charge of lysine residues, weakening the interaction between histones and negatively charged DNA, which typically leads to a more open chromatin structure that facilitates transcriptional activation . Specifically, K36 acetylation on H2A has been associated with transcriptionally active regions and may play roles in DNA repair processes similar to other histone acetylation marks. This modification is catalyzed by histone acetyltransferases such as CBP/p300, as observed with the related histone H2AX variant . The biological significance of this specific modification extends to potential roles in development, cell cycle regulation, and disease processes, particularly in cancer where alterations in histone modification patterns are frequently observed. Understanding the precise function of H2A K36 acetylation contributes to our broader knowledge of epigenetic regulation mechanisms.

How should I optimize the Acetyl-HIST1H2AG (K36) Antibody for Western blotting applications?

Optimization of the Acetyl-HIST1H2AG (K36) Antibody for Western blotting requires careful attention to several experimental parameters to achieve specific detection and minimize background. Begin with proper sample preparation by extracting histones using an acid extraction protocol, which efficiently isolates histones while preserving their post-translational modifications. For histone samples, use 15-18% SDS-PAGE gels to achieve proper resolution of these relatively small proteins (approximately 14-18 kDa) . Initial antibody dilution should be tested in a range from 1:500 to 1:2000, with overnight incubation at 4°C to ensure adequate binding to the target epitope. Including appropriate positive controls (such as cells treated with histone deacetylase inhibitors to increase global acetylation levels) and negative controls (such as samples treated with histone acetyltransferase inhibitors) will help validate the specificity of the detected signals. Blocking solutions containing 5% BSA rather than milk are often preferred for phospho-specific and acetyl-specific antibodies, as milk can contain phosphatases and other enzymes that might interfere with detection of modified proteins. Signal detection can be optimized using enhanced chemiluminescence systems with varying exposure times to identify the optimal signal-to-noise ratio.

What are the critical steps for successful chromatin immunoprecipitation (ChIP) using the Acetyl-HIST1H2AG (K36) Antibody?

Successful chromatin immunoprecipitation using the Acetyl-HIST1H2AG (K36) Antibody requires meticulous attention to several critical steps in the protocol. First, optimal crosslinking conditions must be established, typically using 1% formaldehyde for 10-15 minutes at room temperature, as over-fixation can mask epitopes while under-fixation may result in poor chromatin preservation . Chromatin shearing is another crucial step, with sonication parameters requiring optimization to produce DNA fragments between 200-500 bp for high-resolution mapping of the modification. For immunoprecipitation with the Acetyl-HIST1H2AG (K36) Antibody, using 2-5 μg of antibody per ChIP reaction is generally recommended, with overnight incubation at 4°C to ensure complete binding. Including appropriate controls is essential: an IgG control from the same species as the primary antibody (rabbit) serves as a negative control, while antibodies against known active histone marks (such as H3K4me3 or H3K27ac) can serve as positive controls for transcriptionally active regions. After immunoprecipitation, thorough washing steps are necessary to reduce background, followed by careful elution, reverse crosslinking, and DNA purification. The purified DNA can then be analyzed by qPCR, next-generation sequencing, or other appropriate methods to identify genomic regions enriched for H2A K36 acetylation.

What sample preparation techniques are recommended for immunofluorescence using this antibody?

Successful immunofluorescence with the Acetyl-HIST1H2AG (K36) Antibody requires careful sample preparation to preserve both cellular architecture and the specific histone modification. Begin with fixation using 4% paraformaldehyde for 15-20 minutes at room temperature, which effectively preserves nuclear structure while maintaining accessibility of nuclear antigens . A permeabilization step using 0.1-0.5% Triton X-100 for 10 minutes is essential for allowing antibody access to nuclear epitopes. For detection of histone modifications, an antigen retrieval step may improve signal intensity, typically performed using a citrate buffer (pH 6.0) with heat treatment. Blocking should be performed using 3-5% BSA or normal serum (from a species different from that of the primary and secondary antibodies) for at least 1 hour to minimize non-specific binding. For primary antibody incubation, a dilution range of 1:100 to 1:500 should be tested, with overnight incubation at 4°C generally yielding optimal results. Multiple washing steps with PBS containing 0.1% Tween-20 are crucial between each step of the protocol to reduce background. For visualization, use appropriate fluorophore-conjugated secondary antibodies and include DAPI or another nuclear counterstain to facilitate visualization of nuclear localization. Mounting media containing anti-fade reagents will help preserve fluorescence for imaging and analysis.

How can the Acetyl-HIST1H2AG (K36) Antibody be used to study the dynamics of histone modifications during cell cycle progression?

The Acetyl-HIST1H2AG (K36) Antibody can be instrumental in studying the dynamic changes in histone H2A acetylation patterns throughout the cell cycle through multiple complementary approaches. Time-course experiments can be designed where cells are synchronized at different cell cycle phases using methods such as double thymidine block (G1/S boundary), nocodazole treatment (M phase), or serum starvation followed by release (G0/G1 transition) . At defined time points after synchronization and release, samples can be collected for immunofluorescence microscopy, which allows visualization of the spatial distribution of H2A K36 acetylation within individual nuclei, potentially revealing phase-specific patterns or foci. Parallel samples can be processed for Western blotting to quantify global changes in H2A K36 acetylation levels throughout the cell cycle. For a more comprehensive analysis, ChIP followed by sequencing (ChIP-seq) can be performed at different cell cycle stages to map genome-wide changes in the distribution of this modification, potentially identifying cell cycle-regulated genes associated with this mark . Flow cytometry combining DNA content staining (with propidium iodide) and immunostaining for acetylated H2A K36 can provide single-cell resolution data correlating the modification levels with specific cell cycle phases. These multi-modal approaches can reveal how this specific histone modification is regulated during cellular division and its potential functional significance in cell cycle progression.

What strategies can be employed to investigate the relationship between HIST1H2AG K36 acetylation and transcriptional regulation?

Investigating the relationship between HIST1H2AG K36 acetylation and transcriptional regulation requires integrative approaches combining epigenomic and transcriptomic analyses. ChIP-seq using the Acetyl-HIST1H2AG (K36) Antibody can map the genome-wide distribution of this modification, which can then be correlated with RNA-seq data from the same cell type to identify associations between K36 acetylation and gene expression levels . For more direct evidence of causality, CRISPR-based epigenome editing can be employed, using catalytically inactive Cas9 (dCas9) fused to histone acetyltransferases (to increase K36 acetylation) or deacetylases (to decrease K36 acetylation) at specific genomic loci, followed by measurement of target gene expression changes. Sequential ChIP (re-ChIP) experiments can identify genomic regions where H2A K36 acetylation co-occurs with other histone modifications or transcription factors, providing insights into the combinatorial histone code. The temporal dynamics of this modification during transcriptional activation can be studied using systems where gene expression can be rapidly induced (e.g., heat shock response, hormone treatment), with samples collected at multiple time points for both ChIP and RNA analysis. Proteomic approaches like RIME (Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins) can identify protein complexes associated with acetylated H2A K36, potentially revealing readers of this modification that mediate its effects on transcription. These comprehensive strategies can elucidate how this specific histone modification contributes to the complex landscape of transcriptional regulation.

How can multiplexed immunofluorescence be optimized to study co-localization of HIST1H2AG K36 acetylation with other histone modifications?

Optimizing multiplexed immunofluorescence for studying co-localization of HIST1H2AG K36 acetylation with other histone modifications requires careful planning to avoid antibody cross-reactivity and spectral overlap. Begin by selecting primary antibodies raised in different host species (e.g., rabbit anti-Acetyl-HIST1H2AG K36, mouse anti-H3K27ac, goat anti-H3K4me3) to allow for simultaneous detection with species-specific secondary antibodies . If multiple rabbit-derived antibodies must be used, consider sequential immunostaining with complete elution of antibodies between rounds or use directly conjugated primary antibodies with distinct fluorophores. The order of antibody application should be optimized, typically starting with the antibody detecting the least abundant epitope, followed by more abundant targets. Fluorophore selection is critical, choosing fluorophores with minimal spectral overlap and appropriate brightness for each target's abundance (e.g., brighter fluorophores for less abundant modifications). Include rigorous controls including single-color staining to assess bleed-through, no-primary antibody controls to evaluate secondary antibody specificity, and peptide competition assays to confirm primary antibody specificity. Advanced imaging techniques such as structured illumination microscopy (SIM) or stochastic optical reconstruction microscopy (STORM) can provide super-resolution beyond the diffraction limit, allowing more precise co-localization analysis. Quantitative co-localization analysis should be performed using specialized software like ImageJ with plugins such as JACoP (Just Another Colocalization Plugin) or Coloc2, calculating metrics such as Pearson's correlation coefficient, Mander's overlap coefficient, or intensity correlation quotient to objectively measure the degree of co-localization between different histone modifications.

What are the common pitfalls in Western blotting with the Acetyl-HIST1H2AG (K36) Antibody and how can they be resolved?

Western blotting with the Acetyl-HIST1H2AG (K36) Antibody can encounter several common pitfalls that may compromise experimental results. One frequent issue is high background signal, which can result from insufficient blocking, inadequate washing, or excessive antibody concentration . This can be resolved by extending the blocking step to 1-2 hours with fresh blocking solution (5% BSA is often superior to milk for acetyl-specific antibodies), implementing more stringent washing with increased duration and detergent concentration, and titrating the antibody to find the optimal dilution (typically starting with 1:1000 and adjusting as needed). Another common problem is weak or absent signal, which may stem from low abundance of the target modification, epitope masking during fixation, or degradation of the acetylation mark during sample preparation. Solutions include using histone deacetylase inhibitors (like sodium butyrate or trichostatin A) during sample collection to preserve acetylation, optimizing extraction protocols to include protease and deacetylase inhibitors, and exploring alternative extraction methods specifically designed for histones such as acid extraction. Cross-reactivity with other acetylated histones can also occur due to sequence similarities, which can be addressed by performing peptide competition assays with both specific and non-specific acetylated peptides to confirm antibody specificity. Multiple bands on Western blots may represent other histone variants or differential post-translational modifications, requiring mass spectrometry validation for definitive identification. Inconsistent results between experiments often stem from variations in cell culture conditions affecting global acetylation levels, which can be standardized by controlling cell density, passage number, and serum lots.

How should researchers interpret and validate ChIP-seq data generated using the Acetyl-HIST1H2AG (K36) Antibody?

Interpreting and validating ChIP-seq data generated with the Acetyl-HIST1H2AG (K36) Antibody requires rigorous quality control measures and comprehensive data analysis. Initially, assess basic sequencing metrics including total read count, mapping rate, and library complexity to ensure adequate coverage and data quality . Examine the proportion of reads in peaks (FRIP score), which should typically exceed 1% for histone modifications, with higher values indicating better signal-to-noise ratios. Validate the specificity of binding by analyzing motif enrichment in peak regions, although histone modifications often show broader patterns rather than sharp peaks associated with transcription factors. Compare the distribution of identified peaks with known genomic features such as promoters, enhancers, gene bodies, and intergenic regions to assess biological relevance, expecting acetylation marks to be enriched at active regulatory elements. Visual inspection of normalized signal tracks at well-characterized genes using genome browsers is essential to confirm expected patterns. Validation of key findings should be performed using orthogonal methods such as ChIP-qPCR at selected genomic loci, comparing enrichment relative to input and IgG controls. Correlation with other datasets, such as RNA-seq, DNase-seq, or ATAC-seq, can provide functional context for the identified regions. For definitive validation, perform spike-in normalization using chromatin from a different species with a constant amount of the target modification to control for technical variations between samples. Compare results with publicly available datasets for similar histone modifications to identify consistent and potentially novel patterns, but be aware that cell type-specific differences may exist.

What controls are essential when working with the Acetyl-HIST1H2AG (K36) Antibody, and how do they help troubleshoot experimental issues?

Implementing robust controls when working with the Acetyl-HIST1H2AG (K36) Antibody is crucial for experimental validity and troubleshooting. For Western blotting and immunostaining applications, positive controls should include samples known to have high levels of H2A K36 acetylation, such as cells treated with histone deacetylase inhibitors like sodium butyrate or trichostatin A, which increase global histone acetylation levels . Negative controls should include samples with reduced acetylation, which can be generated by treating cells with histone acetyltransferase inhibitors such as C646 (p300/CBP inhibitor). For all applications, technical controls should include a no-primary antibody condition to assess secondary antibody specificity and background. Peptide competition assays, where the antibody is pre-incubated with excess acetylated peptide matching the target epitope before use in the experiment, serve as critical specificity controls - signal reduction confirms antibody specificity. For ChIP experiments, input DNA (a small portion of chromatin saved before immunoprecipitation) serves as a crucial control for normalization, while immunoprecipitation with isotype-matched IgG evaluates non-specific binding. When troubleshooting weak signals, controls with known high acetylation can confirm whether the issue is biological (low acetylation in the sample) or technical (antibody or protocol problems). For high background problems, comparing results between specific antibody, IgG control, and no-antibody conditions can help identify the source of non-specific binding. These comprehensive controls help distinguish true biological signals from artifacts and provide reference points for optimizing experimental conditions.

How does HIST1H2AG K36 acetylation interact with other histone modifications in the context of the histone code?

HIST1H2AG K36 acetylation participates in the complex language of the histone code through various interactions with other post-translational modifications that collectively regulate chromatin structure and function. Histone modifications exhibit both cis and trans interactions, where modifications on the same histone tail (cis) or different histones (trans) can influence each other through various mechanisms . For example, acetylation at one lysine residue can increase the probability of acetylation at nearby residues by recruiting histone acetyltransferases or by altering the local chromatin structure to make adjacent sites more accessible. The relationship between H2A K36 acetylation and other common histone marks like H3K4 methylation (associated with active promoters) or H3K27 acetylation (marking active enhancers) may create specific combinatorial patterns that define distinct functional genomic regions. Some modifications exhibit antagonistic relationships where one modification prevents another, such as the mutual exclusivity often observed between acetylation and methylation at the same lysine residue. The biological interpretation of H2A K36 acetylation likely depends on its genomic context and co-occurring modifications, potentially signaling different outcomes when present in promoters versus gene bodies or enhancers. The complex interplay between H2A K36 acetylation and other modifications is mediated by "reader" proteins containing specialized domains (such as bromodomains for acetylated lysines) that recognize specific modifications or combinatorial patterns and recruit additional regulatory complexes to affect downstream processes like transcription, replication, or DNA repair.

What role does HIST1H2AG K36 acetylation play in DNA damage response and genomic stability?

The role of HIST1H2AG K36 acetylation in DNA damage response (DDR) and genomic stability represents an important area of investigation, especially given the established functions of histone modifications in DNA repair processes. Histone acetylation generally facilitates chromatin relaxation, potentially allowing repair factors greater access to damaged DNA, with site-specific acetylation marks often serving as signals to recruit specific DNA repair machinery . While specific research on HIST1H2AG K36 acetylation in DDR is limited, studies on the related histone variant H2AX have demonstrated that K36 acetylation by CBP/p300 acetyltransferases occurs in response to DNA damage. This modification may work in concert with the well-characterized γH2AX (phosphorylation at serine 139), which serves as a critical marker of DNA double-strand breaks and facilitates recruitment of repair factors. The potential crosstalk between acetylation and phosphorylation on histone H2A variants suggests a complex signaling mechanism that fine-tunes the DNA damage response. Acetylation at specific lysine residues can influence the kinetics of DNA repair by affecting chromatin compaction or by creating binding sites for proteins containing bromodomains, which specifically recognize acetylated lysines. In cancer cells, where genomic instability is a hallmark feature, alterations in histone acetylation patterns, potentially including HIST1H2AG K36 acetylation, may contribute to impaired DNA repair mechanisms and increased mutation rates. Understanding the specific functions of this modification in DDR could provide insights into mechanisms of cancer development and potential therapeutic vulnerabilities, particularly in cancers with defective DNA repair pathways.

How might emerging technologies enhance our understanding of HIST1H2AG K36 acetylation function and regulation?

Emerging technologies offer transformative potential for advancing our understanding of HIST1H2AG K36 acetylation's function and regulatory mechanisms. Single-cell epigenomic techniques, including single-cell ChIP-seq and CUT&Tag, will enable researchers to map this modification at unprecedented resolution, revealing cell-to-cell heterogeneity that may be masked in bulk population analyses . These approaches could uncover how this modification varies across cell types or states within complex tissues or tumors. Live-cell imaging of histone modifications, made possible through the development of genetically encoded sensors for specific histone marks, could allow real-time visualization of dynamic changes in H2A K36 acetylation during processes like cell division, differentiation, or response to environmental stimuli. CRISPR-based epigenome editing technologies using catalytically inactive Cas9 (dCas9) fused to histone acetyltransferases or deacetylases enable precise manipulation of this modification at specific genomic loci, facilitating direct testing of its functional consequences on chromatin structure and gene expression. Mass spectrometry-based proteomics approaches with improved sensitivity can identify proteins that specifically recognize, establish, or remove this modification, illuminating the molecular mechanisms through which it exerts its effects. Spatial transcriptomics and in situ sequencing technologies, when combined with immunofluorescence for histone modifications, could reveal the spatial organization of genome regions marked by H2A K36 acetylation within the nucleus, potentially connecting nuclear architecture to gene regulation. These technological advances, especially when used in combination, promise to provide a more comprehensive and nuanced understanding of how this specific histone modification contributes to epigenetic regulation.

What are the potential applications of HIST1H2AG K36 acetylation in personalized medicine and targeted therapeutics?

The study of HIST1H2AG K36 acetylation holds promising implications for personalized medicine and targeted therapeutics, particularly in cancer treatment. Profiling patients' tumors for specific histone modification patterns, including H2A K36 acetylation, could potentially serve as a biomarker for disease stratification, helping to classify tumors beyond traditional histopathological categories and predict response to specific therapeutic regimens . The enzymes responsible for adding (histone acetyltransferases) or removing (histone deacetylases) this modification represent potential drug targets, with several HDAC inhibitors already approved for certain cancers and more specific inhibitors in development. Understanding the genomic regions and genes affected by this modification could identify downstream targets for therapeutic intervention that may be more amenable to drug development than directly targeting the epigenetic marks themselves. Combining epigenetic profiling with other omics approaches (genomics, transcriptomics, proteomics) could create comprehensive tumor signatures that guide personalized treatment strategies, potentially revealing synthetic lethal interactions where tumors with specific epigenetic profiles show vulnerability to particular drugs. Monitoring changes in H2A K36 acetylation patterns during treatment could serve as a pharmacodynamic marker to assess drug efficacy and development of resistance mechanisms. In the realm of drug discovery, high-throughput screening platforms incorporating readouts for specific histone modifications could identify novel compounds that selectively modulate H2A K36 acetylation or target the proteins that recognize this modification, potentially expanding the epigenetic therapy arsenal beyond current HDAC inhibitors to more targeted approaches with fewer side effects.

What experimental approaches can address the causality between HIST1H2AG K36 acetylation and specific cellular processes?

Establishing causality between HIST1H2AG K36 acetylation and specific cellular processes requires sophisticated experimental approaches that go beyond correlative observations. CRISPR-based epigenome editing represents a powerful strategy, wherein catalytically inactive Cas9 (dCas9) fused to either histone acetyltransferases (to increase acetylation) or deacetylases (to decrease acetylation) can be targeted to specific genomic loci using appropriate guide RNAs . This approach allows researchers to directly manipulate H2A K36 acetylation at individual genes or regulatory elements and observe the resulting phenotypic effects on processes such as transcription, replication, or DNA repair. Complementary to targeted approaches, global manipulation of this modification can be achieved through chemical genetics, using engineered histone acetyltransferases or deacetylases that can be selectively activated or inhibited by small molecules, allowing temporal control over modification levels. Genetic approaches creating mutations that either mimic acetylation (lysine to glutamine substitution) or prevent it (lysine to arginine substitution) at position 36 of HIST1H2AG can provide insights into the functional consequences of this modification, though careful control experiments are needed to distinguish direct effects from potential structural changes caused by the mutations. Proteomic approaches such as BioID or APEX proximity labeling can identify proteins that associate with chromatin regions marked by H2A K36 acetylation, potentially revealing the effector molecules that mediate its downstream effects. Time-resolved experiments combining inducible systems with sequential ChIP, RNA-seq, and phenotypic assays can establish the temporal sequence of events following changes in H2A K36 acetylation, which is essential for distinguishing cause from consequence. These multi-faceted approaches, especially when applied in combination, can establish causal relationships between this specific histone modification and its biological functions.