Acetyl-HIST1H3A (K36) Antibody

What is Acetyl-HIST1H3A (K36) and why is it significant in epigenetic research?

Acetyl-HIST1H3A (K36) refers to the acetylation of lysine 36 on histone H3.1, a specific post-translational modification with important regulatory functions. This modification plays a critical role in gene regulation and chromatin remodeling, making it an essential focus in epigenetics research . Acetylation at specific lysine residues on histones can modify chromatin structure, influencing DNA accessibility and subsequent transcriptional activity. Specifically, acetylation of histone H3 at K36 has been implicated in various biological processes, including transcriptional activation, DNA replication, and DNA repair .

The significance of this modification extends to multiple cellular processes, as altered histone acetylation patterns are associated with various disease states, particularly cancers. Understanding the dynamics of H3K36 acetylation provides valuable insights into the mechanisms underlying gene regulation in both normal and pathological conditions .

How does Acetyl-HIST1H3A (K36) differ from methylated H3K36?

While both modifications occur at the same amino acid residue (lysine 36 of histone H3), acetylation and methylation serve distinct biological functions and are regulated by different enzyme systems:

A key distinction is in their regulation - methylation of H3K36 is mediated through a trans-histone regulatory mechanism involving Set2 and histone H4, particularly residue K44 . This trans-histone pathway does not appear to be shared with acetylation regulation. Additionally, H3K36 methylation can recruit specific protein complexes like the Rpd3S histone deacetylase complex through recognition by the chromodomain of Eaf3 and the plant homeobox domain of Rco1 .

What applications are Acetyl-HIST1H3A (K36) antibodies validated for?

Acetyl-HIST1H3A (K36) antibodies have been validated for multiple experimental applications in epigenetic research:

• Western Blot (WB) - For detecting and quantifying acetylated H3K36 in protein lysates, typically used at dilutions of 1:500-1:1000

• Immunofluorescence/Immunocytochemistry (IF/ICC) - For visualizing cellular localization of acetylated H3K36, with recommended dilutions of 1:50-1:100

• Enzyme-Linked Immunosorbent Assay (ELISA) - For quantitative detection of acetylated H3K36 in solution

• Chromatin Immunoprecipitation (ChIP) - For identifying genomic regions enriched with acetylated H3K36, a crucial technique for understanding the distribution of this modification across the genome

The selection of the appropriate application depends on the specific research question, with ChIP being particularly valuable for mapping genomic locations of this modification in relation to gene structure and expression status .

How should Acetyl-HIST1H3A (K36) antibodies be validated for ChIP experiments?

Proper validation of Acetyl-HIST1H3A (K36) antibodies for ChIP experiments requires a systematic approach to ensure specificity and reproducibility:

• Antibody specificity testing: Before ChIP experiments, validate antibody specificity through:

  • Western blot analysis using nuclear extracts to confirm single band detection at the expected molecular weight (approximately 17kDa observed for histone H3)

  • Peptide competition assays using the acetylated and unacetylated peptides

  • Testing on samples with known acetylation status (e.g., cells treated with histone deacetylase inhibitors)

• Positive controls: Include positive control regions known to be enriched for H3K36 acetylation, such as actively transcribed genes

• Negative controls:

  • IgG control immunoprecipitation to assess non-specific binding

  • Regions known to lack H3K36 acetylation

  • Samples from cells where H3K36 acetylation has been experimentally depleted

• Cross-validation: Compare ChIP results with other acetylation-specific antibodies or with orthogonal techniques like CUT&RUN or CUT&Tag

• Sequential ChIP (ChIP-reChIP): Perform sequential immunoprecipitation with antibodies against general H3 and then acetyl-H3K36 to confirm specificity

The validation method should include qPCR analysis of enriched regions and potentially genome-wide sequencing to establish a comprehensive profile of the modification, similar to approaches used for methylated H3K36 .

What are the optimal sample preparation techniques for detecting Acetyl-HIST1H3A (K36)?

Optimal sample preparation is crucial for reliable detection of Acetyl-HIST1H3A (K36) and varies by application:

For Western Blot analysis:

• Harvest cells in the exponential growth phase (typically 70-80% confluent)

• Extract histones using acid extraction method (0.2N HCl or 0.4N H2SO4) to effectively isolate histones

• Include histone deacetylase inhibitors (e.g., sodium butyrate, trichostatin A) in all buffers

• Use SDS-PAGE with 15-18% polyacrylamide gels for optimal histone separation

• Transfer proteins to PVDF membrane (preferred over nitrocellulose for histone proteins)

• Block with 5% BSA rather than milk proteins (which contain phosphatases that may affect results)

• Dilute primary antibody as recommended (1:500-1:1000)

For ChIP experiments:

• Optimize crosslinking conditions (typically 1% formaldehyde for 10 minutes at room temperature)

• Use sonication conditions that produce chromatin fragments of 200-500 bp

• Include protease inhibitors, deacetylase inhibitors, and phosphatase inhibitors in all buffers

• Pre-clear chromatin with protein A/G beads before immunoprecipitation

• Use 2-5 μg antibody per IP reaction

• Include input control (typically 5-10% of starting chromatin)

• Include IgG negative control

• Reverse crosslinks completely (typically 65°C overnight)

The use of positive control cell lines with known H3K36 acetylation patterns, such as HeLa or NIH/3T3 cells, is recommended for initial optimization experiments .

How should researchers interpret conflicting results between Acetyl-H3K36 and Methyl-H3K36 patterns?

When researchers encounter conflicting patterns between acetylation and methylation at H3K36, systematic analysis is required:

• Confirm antibody specificity: Verify that antibodies are truly specific for acetylation or methylation states through peptide competition assays and western blot validation.

• Consider temporal dynamics: Acetylation and methylation may occur in a sequential manner during transcription or other processes, so the timing of sample collection may influence results.

• Examine enzyme regulation: Analyze the presence and activity of writers (acetyltransferases, methyltransferases), erasers (deacetylases, demethylases), and readers of each modification in your experimental system.

• Address potential mutual exclusivity: Since acetylation and methylation target the same lysine residue, these modifications are mutually exclusive on a single histone molecule. Conflicting signals may represent different subpopulations of histones.

• Evaluate genomic resolution: The resolution of your detection method may influence interpretation - ChIP-seq provides genome-wide patterns while ChIP-qPCR examines specific loci.

• Consider trans-histone regulation: As demonstrated in yeast, H3K36 methylation is regulated by a trans-histone mechanism involving histone H4 . Similar mechanisms may influence acetylation patterns.

• Analyze biological context: Different cell types, developmental stages, or experimental conditions may result in different modification patterns.

It's important to note that H3K36 methylation has been shown to recruit histone deacetylase complexes that remove acetyl groups from histones within transcribed regions . This functional interplay between methylation and acetylation may explain some seemingly conflicting results.

How does Acetyl-H3K36 interact with other histone modifications in the histone code?

Acetyl-H3K36 functions within a complex network of histone modifications that collectively regulate chromatin structure and gene expression:

• Crosstalk with H3K36 methylation: Since acetylation and methylation cannot occur simultaneously on the same K36 residue, these modifications may antagonize each other. H3K36 methylation can recruit the Rpd3S histone deacetylase complex, potentially affecting acetylation patterns at other residues .

• Coordination with H3K4 modifications: While H3K36 methylation and H3K4 methylation have distinct roles, they can work in concert to regulate transcription. Research indicates that Set2-dependent H3K36 methylation, but not Set1-dependent H3K4 methylation, stimulates the association of certain factors (like Rad26p) with coding sequences of active genes .

• Relationship with other acetylation marks: H3K36 acetylation likely functions alongside other acetylation marks on histones H3 and H4. The precise patterns and temporal sequences of multiple acetylation events can determine functional outcomes.

• Trans-histone regulation mechanisms: Evidence from studies on H3K36 methylation suggests that modifications on one histone can influence modifications on another. For example, specific residues in histone H4 (particularly K44) are critical for proper H3K36 methylation . Similar mechanisms may exist for acetylation regulation.

• Role in transcriptional elongation: Both H3K36 methylation and acetylation have connections to transcriptional elongation, potentially with different roles in regulating RNA polymerase II progression along genes.

Understanding these interactions requires multi-modal approaches that can simultaneously detect multiple modifications, such as sequential ChIP (ChIP-reChIP) or mass spectrometry-based proteomics.

What are the mechanisms regulating Acetyl-H3K36 during the cell cycle?

The regulation of H3K36 acetylation throughout the cell cycle involves complex mechanisms that coordinate this modification with DNA replication, chromatin assembly, and gene expression:

• Cell cycle-specific acetyltransferases and deacetylases: Different HATs and HDACs may be active during specific cell cycle phases, regulating the establishment and removal of H3K36 acetylation.

• Chromatin assembly during S phase: During DNA replication, newly synthesized histones are incorporated into chromatin. These histones undergo a specific sequence of modifications, including acetylation, before being assembled into nucleosomes. H3K36 acetylation may be part of this pre-deposition modification pattern.

• Coordination with transcriptional programs: Cell cycle-regulated genes show dynamic changes in histone modifications, including H3K36 modifications. The acetylation state may correspond to the transcriptional activity of specific gene sets during different cell cycle phases.

• Integration with DNA damage responses: Since H3K36 modifications are implicated in DNA repair processes, its acetylation state may change in response to DNA damage occurring during replication.

• Relationship with kinetochore assembly: Histone modifications, including acetylation, can influence centromere and kinetochore assembly during mitosis. The specific role of H3K36 acetylation in this process remains to be fully characterized.

Research methodologies to study these dynamics should include:

• Synchronization of cells at different cell cycle stages

• ChIP-seq analysis across the cell cycle

• Immunofluorescence microscopy to track modification patterns during mitosis

• Proteomic analysis of histone modifications at different cell cycle phases

• Functional studies using acetyltransferase or deacetylase inhibitors at specific cell cycle stages

How can researchers distinguish between the roles of Acetyl-H3K36 and H3K36me3 in transcriptional regulation?

Distinguishing between the roles of acetylation and trimethylation at H3K36 in transcriptional regulation requires sophisticated experimental approaches:

• Genomic distribution analysis:

  • Perform ChIP-seq with specific antibodies for each modification

  • Compare distribution patterns relative to transcription start sites, gene bodies, enhancers, and other genomic features

  • Analyze co-occurrence or mutual exclusivity patterns

• Temporal dynamics studies:

  • Conduct time-course experiments during transcriptional activation or repression

  • Use techniques like metabolic labeling of histones to track newly deposited modifications

  • Apply rapid immunoprecipitation techniques to capture transient states

• Enzyme manipulation experiments:

  • Selectively inhibit or deplete specific acetyltransferases vs. methyltransferases

  • Analyze resulting effects on transcription using RNA-seq

  • Employ rapid enzyme degradation systems (e.g., auxin-inducible degron) for temporal control

• Reader protein identification:

  • Perform affinity purification with modified peptides to identify specific readers

  • Use BioID or APEX proximity labeling to identify proteins associated with each modification in living cells

  • Analyze differential recruitment of transcriptional machinery

• High-resolution techniques:

  • Apply techniques like CUT&RUN or CUT&Tag for improved signal-to-noise ratio

  • Use single-molecule imaging to track modification dynamics in living cells

  • Employ nascent RNA sequencing (e.g., NET-seq, PRO-seq) to directly correlate modifications with transcriptional activity

Evidence from research on H3K36 methylation indicates that this modification is involved in preventing spurious intragenic transcription by recruiting histone deacetylases , while acetylation may play a more direct role in promoting transcription. The presence of H3K36me3 at the coding sequence of active genes, such as GAL1 in yeast, suggests a role in regulation of active transcription .

What experimental approaches can address the relationship between Acetyl-H3K36 and DNA repair mechanisms?

Investigating the relationship between Acetyl-H3K36 and DNA repair requires methodologies that can link this specific histone modification to repair processes:

• DNA damage induction studies:

  • Create defined DNA lesions using site-specific nucleases (e.g., CRISPR-Cas9, I-SceI)

  • Apply different DNA damaging agents (UV, ionizing radiation, chemicals) to induce specific types of damage

  • Track Acetyl-H3K36 patterns before and after damage induction using ChIP

• Repair kinetics analysis:

  • Perform time-course ChIP experiments following DNA damage

  • Combine with γH2AX ChIP to correlate with known damage markers

  • Use repair-specific antibodies to track resolution of damage alongside histone modification changes

• Repair pathway dissection:

  • Utilize cells deficient in specific repair pathways (homologous recombination, non-homologous end joining, nucleotide excision repair)

  • Analyze how pathway deficiencies affect Acetyl-H3K36 patterns

  • Compare with known methylation patterns, as H3K36me3 has established roles in homologous recombination repair

• Enzyme manipulation approaches:

  • Deplete or inhibit acetyltransferases/deacetylases that target H3K36

  • Assess impact on repair efficiency using reporter assays

  • Complement with methyltransferase studies (e.g., SET2 deletion) to compare with known effects

• Protein recruitment studies:

  • Identify repair factors that recognize or are influenced by Acetyl-H3K36

  • Perform interaction studies using modified peptides as bait

  • Compare with factors known to interact with methylated H3K36

Research has shown that H3K36 methylation stimulates the association of Rad26p with coding sequences of active genes , suggesting roles in transcription-coupled repair. Similar experimental approaches could reveal whether Acetyl-H3K36 has distinct or overlapping functions in DNA repair processes.

What are common pitfalls in ChIP experiments using Acetyl-H3K36 antibodies and how can they be addressed?

ChIP experiments with Acetyl-H3K36 antibodies can encounter several challenges that require specific troubleshooting approaches:

• Low signal-to-noise ratio:

  • Problem: High background or weak specific signal

  • Solutions:

    • Optimize antibody concentration (typically start with 2-5 μg per reaction)

    • Increase washing stringency (adjust salt concentration)

    • Use alternative blocking agents (BSA vs. milk proteins)

    • Include additional pre-clearing steps

• Cross-reactivity issues:

  • Problem: Antibody recognizes other acetylated lysines

  • Solutions:

    • Perform peptide competition assays with specific acetylated peptides

    • Test antibody specificity by Western blot on samples with HDAC inhibitors

    • Compare results with multiple antibodies from different vendors

    • Validate with mass spectrometry

• Deacetylation during sample processing:

  • Problem: Loss of acetylation signal during experiment

  • Solutions:

    • Add HDAC inhibitors (sodium butyrate, trichostatin A) to all buffers

    • Keep samples cold throughout processing

    • Minimize time between sample collection and fixation

    • Use freshly prepared buffers

• Inefficient chromatin shearing:

  • Problem: Poor fragmentation leads to high background

  • Solutions:

    • Optimize sonication conditions for your specific cell type

    • Verify fragment size by agarose gel electrophoresis

    • Consider alternative fragmentation methods (enzymatic digestion)

    • Optimize crosslinking time (excessive crosslinking can impede sonication)

• Antibody batch variability:

  • Problem: Inconsistent results between experiments

  • Solutions:

    • Purchase antibodies in larger lots when possible

    • Test each new lot against previous lots

    • Include standard positive controls in each experiment

    • Consider monoclonal antibodies for greater consistency

• PCR amplification bias:

  • Problem: Certain regions amplify preferentially in qPCR

  • Solutions:

    • Design multiple primer pairs for regions of interest

    • Optimize PCR conditions for each primer set

    • Consider sequencing-based readout instead of qPCR

When using cell lines for positive controls, HeLa and NIH/3T3 have been validated for Acetyl-H3K36 detection .

How should researchers optimize antibody concentration for different applications?

Optimizing antibody concentration is crucial for obtaining specific signals while minimizing background across different applications:

Western Blot Optimization:

• Start with the manufacturer's recommended dilution range (typically 1:500-1:1000 for Acetyl-H3K36 antibodies)

• Perform a dilution series experiment (e.g., 1:250, 1:500, 1:1000, 1:2000)

• Assess signal-to-noise ratio at each concentration

• Consider exposure time optimization alongside antibody dilution

• Include positive controls (histone extracts from cells treated with HDAC inhibitors)

• For rabbit polyclonal antibodies, secondary antibody optimization may also be necessary

Immunofluorescence/Immunocytochemistry Optimization:

• Begin with recommended dilutions (approximately 1:50-1:100)

• Test a range of concentrations in a dilution series

• Evaluate signal intensity versus background fluorescence

• Consider fixation method effects (paraformaldehyde versus methanol)

• Optimize permeabilization conditions

• Test alternative blocking solutions (BSA, normal serum, commercial blockers)

ChIP Optimization:

• Start with 2-5 μg antibody per IP reaction

• Perform antibody titration experiments (e.g., 1, 2, 5, 10 μg)

• Test different chromatin amounts with fixed antibody concentration

• Compare enrichment at positive control regions versus negative regions

• Calculate signal-to-input ratios at each concentration

• Consider the chromatin concentration effect on optimal antibody amount

ELISA Optimization:

• Create a standard curve with known concentrations of acetylated peptide

• Test primary antibody in 2-fold dilution series

• Optimize secondary antibody concentration independently

• Consider plate coating conditions and blocking reagents

• Optimize incubation times and temperatures

For all applications, document optimization experiments thoroughly and maintain consistent conditions for comparable results across experiments.

What strategies can researchers use when Acetyl-H3K36 signals appear inconsistent between replicates?

When faced with inconsistent Acetyl-H3K36 signals between experimental replicates, researchers should implement a systematic troubleshooting approach:

• Standardize sample preparation:

  • Ensure consistent cell culture conditions (passage number, confluence, media batch)

  • Synchronize cells if studying cell cycle-dependent processes

  • Standardize extraction protocols with precise timing and temperature control

  • Use internal controls to normalize for extraction efficiency

• Control for technical variables:

  • Use the same antibody lot across experiments

  • Prepare fresh buffers for each experiment

  • Standardize incubation times and temperatures

  • Implement consistent washing protocols

  • For ChIP, ensure consistent sonication efficiency across samples

• Quantify and account for sources of variation:

  • Include spike-in controls (e.g., Drosophila chromatin for mammalian ChIP)

  • Use normalization methods appropriate for your application

  • For Western blot, normalize to total H3 on the same membrane

  • For ChIP-qPCR, normalize to input and internal control regions

• Implement robust experimental design:

  • Increase biological replicate number

  • Randomize sample processing order

  • Conduct replicate experiments on different days

  • Consider technical replicates within each biological replicate

• Optimize detection methods:

  • For Western blot, try different exposure times and detection methods

  • For ChIP, test alternative PCR primers or sequencing approaches

  • For microscopy, optimize image acquisition settings

• Apply appropriate statistical analyses:

  • Use statistical tests appropriate for your experimental design

  • Consider power analysis to determine adequate sample size

  • Apply normalization methods to account for technical variation

  • Identify and handle outliers appropriately

• Validate with orthogonal approaches:

  • Confirm findings with alternative antibodies

  • Use complementary techniques (e.g., mass spectrometry)

  • Consider alternative assays that measure the same modification

Remember that biological variability in histone modifications is expected as they respond to cellular signaling and environmental conditions. Distinguishing this natural variability from technical inconsistency is crucial for proper interpretation.

What are emerging research directions for Acetyl-H3K36 studies?

Several promising research directions are emerging in the field of Acetyl-H3K36 studies:

• Single-cell epigenomics: Developing methods to detect Acetyl-H3K36 at single-cell resolution will reveal cell-to-cell heterogeneity and developmental trajectories in complex tissues. This may involve adapting single-cell ChIP-seq, CUT&Tag, or mass cytometry approaches for Acetyl-H3K36 detection.

• Acetylation dynamics and turnover: Implementing metabolic labeling approaches (e.g., SILAC, CATCH-IT) to study the kinetics of Acetyl-H3K36 deposition and removal in response to various stimuli and during different cellular processes.

• Targeted modulation technologies: Developing tools for site-specific modulation of Acetyl-H3K36, such as targeted histone acetyltransferases or deacetylases using CRISPR-dCas9 fusion proteins, to establish causal relationships with gene expression.

• Crosstalk with other chromatin features: Investigating the interplay between Acetyl-H3K36 and other features like DNA methylation, chromatin accessibility, and three-dimensional genome organization through multi-omics approaches.

• Computational modeling: Developing predictive models that integrate multiple histone modifications, including Acetyl-H3K36, to predict functional outcomes and gene expression patterns.

• Therapeutic targeting: Exploring the potential of drugs targeting writers, erasers, or readers of Acetyl-H3K36 for treating diseases with epigenetic dysregulation, particularly cancers.

• Evolutionary perspectives: Comparative studies across species to understand the conservation and divergence of Acetyl-H3K36 functions in different organisms.

These emerging directions build upon the foundational understanding that histone modifications like Acetyl-H3K36 are critical for gene regulation and chromatin remodeling , while exploring new technological and conceptual frontiers.

How does understanding Acetyl-H3K36 contribute to broader epigenetic research?

Understanding Acetyl-H3K36 contributes significantly to the broader landscape of epigenetic research in several ways:

• Expanding the histone code: Acetyl-H3K36 adds an important component to the histone code hypothesis, which proposes that combinations of histone modifications create a complex language that regulates genome function. The interplay between this acetylation mark and other modifications, such as methylation at the same residue, illustrates the complexity and specificity of chromatin regulation .

• Bridging transcription and chromatin structure: Research on H3K36 modifications has revealed critical connections between transcriptional processes and chromatin organization. Both acetylation and methylation at H3K36 appear to be linked to active transcription, but likely with distinct regulatory functions .

• Demonstrating trans-histone regulation: Studies showing that histone H4 influences H3K36 methylation through a trans-histone mechanism highlight the interconnected nature of the nucleosome and how modifications on one histone can affect modifications on another . Similar principles may apply to acetylation regulation.

• Connecting epigenetic modifications to cellular processes: H3K36 modifications have been linked to diverse processes including transcription elongation, DNA repair, and replication, demonstrating how epigenetic marks serve as integration points for multiple nuclear functions .

• Providing research tools and methodologies: The development and characterization of specific antibodies against Acetyl-H3K36 has enabled researchers to study this modification in various contexts, contributing to the broader technical toolkit available for epigenetic research .

• Illustrating context-dependent functions: Research on H3K36 modifications illustrates how the same amino acid residue can carry different modifications with distinct functions, reinforcing the importance of studying histone modifications in their specific biological contexts.