Mono-methyl-HIST1H3A (K36) Antibody

What is Mono-methyl-HIST1H3A (K36) and what biological significance does it have?

Mono-methyl-HIST1H3A (K36) refers to histone H3 that is mono-methylated at lysine 36. Histone H3 is a core component of nucleosomes, which wrap and compact DNA into chromatin, limiting DNA accessibility to cellular machineries that require DNA as a template. H3K36 methylation plays a central role in transcription regulation, DNA repair, DNA replication, and chromosomal stability . Specifically, H3K36 methylation is closely associated with RNA polymerase II elongation during transcription, marking actively transcribed regions of the genome .

The mono-methylation state at K36 represents one of three possible methylation states at this position (mono-, di-, or tri-methylation). Each methylation state may have distinct biological functions and is regulated by specific methyltransferases, with Set2 being a key enzyme responsible for K36 methylation in yeast models . This modification is part of the complex "histone code" that regulates DNA accessibility through post-translational modifications and nucleosome remodeling .

What detection methods can be used with Mono-methyl-HIST1H3A (K36) antibody?

Mono-methyl-HIST1H3A (K36) antibodies can be utilized in multiple experimental applications:

• Western Blot (WB): Effective for detecting H3K36me1 in histone preparations and cell lysates, typically using dilutions around 1/1000. The predicted band size for histone H3 is approximately 15 kDa .

• Immunocytochemistry/Immunofluorescence (ICC/IF): Used for cellular localization studies, typically at dilutions around 1/100. This method allows visualization of the nuclear distribution pattern of H3K36me1 .

• Dot Blot: Useful for assessing antibody specificity against various modified peptides. This approach can distinguish between unmodified K36, K36me1, K36me2, K36me3, and K36ac modifications .

• Enzyme-Linked Immunosorbent Assay (ELISA): Provides quantitative measurement of H3K36me1 levels in samples .

• Chromatin Immunoprecipitation (ChIP): While not explicitly mentioned in the search results, H3K36 methylation antibodies are commonly used in ChIP assays to identify genomic regions enriched with this modification, particularly in transcribed regions of genes .

How conserved is K36 methylation across different species?

H3K36 methylation is highly conserved across eukaryotes, suggesting its fundamental importance in chromatin regulation. Research has demonstrated the presence of K36 methylation in diverse organisms including budding yeast (Saccharomyces cerevisiae), fission yeast (Schizosaccharomyces pombe), Tetrahymena thermophila, chicken, and humans .

While the modification is conserved, the relative abundance of K36 methylation can vary between species. For example, K36 dimethylation appears less abundant in Tetrahymena thermophila compared to yeast, chicken, and humans. This difference might be partly attributed to slight variations in the amino acid sequence surrounding K36; in yeast, chicken, and humans, K36 is preceded by valine (GGVKKPH), while in Tetrahymena thermophila H3.1, it is preceded by isoleucine (GGIKKPH) .

The conservation of Set2-mediated K36 methylation across species, particularly between budding and fission yeasts, further underscores the evolutionary importance of this modification in transcription elongation processes .

How can researchers distinguish between different methylation states (mono-, di-, tri-) at H3K36 in experimental settings?

Distinguishing between different methylation states at H3K36 requires careful experimental approach and antibody selection:

Antibody Specificity Validation:

• Dot blot analysis with peptide arrays containing unmodified K36, K36me1, K36me2, K36me3, and other modifications (like K36ac) is essential to confirm antibody specificity .

• In a typical dot blot, researchers should load increasing amounts of peptide (e.g., 0.3, 0.6, 1, 3, 6, and 10 picomoles) to establish detection sensitivity and specificity thresholds .

Cross-Reactivity Testing:

• Western blots using recombinant histone proteins or synthetic peptides with defined methylation states can help determine antibody cross-reactivity.

• Cold HMT (histone methyltransferase) assays using unlabeled SAM cofactor followed by Western blot analysis with methylation-specific antibodies can verify specificity for particular methylation states .

Mass Spectrometry Validation:

• For definitive characterization of methylation states, mass spectrometry analysis should be employed to confirm the presence and relative abundance of mono-, di-, and tri-methylation.

• This approach can overcome limitations of antibody-based detection methods and provide quantitative information about different methylation states .

Sequential ChIP:

• For genomic studies, sequential ChIP (re-ChIP) using antibodies specific for different methylation states can help identify regions with overlapping or distinct patterns of H3K36 methylation.

What are the methodological considerations for histone methyltransferase assays when studying H3K36 methylation?

When conducting histone methyltransferase (HMT) assays to study H3K36 methylation, researchers should consider several methodological aspects:

Substrate Selection:

• Different substrates exhibit varying efficiencies in HMT assays. Options include recombinant H3, core histones, oligonucleosomes, and H3 synthetic peptides .

• Nucleosomal substrates often provide higher activity for enzymes like Set2, which preferentially methylate nucleosome-bound H3 .

Reaction Conditions:

• Standard reaction conditions typically include methyltransferase buffer (50 mM Tris, pH 9.0, 10% glycerol, protease inhibitors) with either radiolabeled ([³H]SAM) or non-radiolabeled S-adenosyl-L-methionine (SAM) as methyl donor .

• Incubation is generally performed at 30°C for approximately 30 minutes .

Detection Methods:

Controls and Specificity Verification:

• Include negative controls (no enzyme) and positive controls (known methyltransferases) in each experiment.

• Verify site specificity using peptides with pre-existing modifications that should block further methylation. For example, a peptide trimethylated at K36 should not be a substrate for K36-specific methyltransferases .

• Test methylation activity against peptides covering different regions of H3 (e.g., residues 1-20 vs. 27-45) to confirm site specificity .

What are the technical considerations when optimizing Western blot protocols for H3K36me1 detection?

Optimizing Western blot protocols for H3K36me1 detection requires attention to several technical considerations:

Sample Preparation:

• Histone extraction methods significantly impact results. For H3K36me1 detection, specialized histone prep protocols are recommended over standard whole-cell lysates .

• Load appropriate amounts of histone preparations (approximately 30 μg for cell line samples) .

Antibody Dilution and Incubation:

• The recommended dilution for anti-H3K36me1 antibodies is typically around 1/1000 for Western blot applications .

• Optimization may be required based on sample type and antibody batch.

Secondary Antibody Selection:

• For quantitative analysis, fluorescent secondary antibodies (e.g., IRDye800TM) provide superior quantification capabilities compared to chemiluminescent detection .

• Typical dilutions for fluorescent secondary antibodies are around 1/10000 .

Controls and Validation:

• Include positive controls (cell lines with known H3K36me1 levels, such as HeLa cells) .

• Include multiple organisms when possible to account for cross-species variations (e.g., human and C. elegans samples) .

• Verify the expected band size (approximately 15 kDa for histone H3) .

Troubleshooting High Background:

• Increase blocking time or concentration of blocking agent.

• Increase washing steps or washing buffer stringency.

• Further dilute primary and/or secondary antibodies.

• Use highly specific antibodies validated for Western blot applications.

How can researchers integrate H3K36 methylation data with other histone modifications to understand the histone code?

Integrating H3K36 methylation data with other histone modifications requires a comprehensive analytical approach:

Multi-Antibody ChIP-seq Studies:

• Perform parallel ChIP-seq experiments using antibodies against H3K36me1, H3K36me2, H3K36me3, and other functionally related modifications (e.g., H3K4me3, H3K27ac, H3K9me3).

• Use sequential ChIP (re-ChIP) to identify genomic regions containing combinations of specific histone marks.

Correlation Analysis:

• Calculate genome-wide correlation coefficients between H3K36 methylation and other histone modifications.

• Generate heatmaps clustering genes based on patterns of multiple histone modifications.

Functional Context Analysis:

• Integrate RNA-seq data to correlate H3K36 methylation patterns with transcriptional activity .

• Analyze H3K36 methylation in relation to gene structure (promoters, gene bodies, intron-exon boundaries).

• Map H3K36 methylation in conjunction with RNA polymerase II occupancy to understand relationships with transcription elongation .

Mass Spectrometry Approaches:

• Use top-down proteomics to identify combinatorial patterns of histone modifications co-occurring on the same histone molecules.

• Quantify relative abundances of different histone modification combinations.

Computational Integration:

• Employ machine learning algorithms to identify patterns and relationships between different histone modifications.

• Use chromatin state models (e.g., ChromHMM) to define functional chromatin states based on combinations of histone marks.

Understanding that H3K36 methylation is primarily associated with transcribed regions and elongation by RNA polymerase II provides important context for interpretation in relation to other histone modifications .

How can researchers troubleshoot inconsistent results when using Mono-methyl-HIST1H3A (K36) antibody?

When facing inconsistent results with Mono-methyl-HIST1H3A (K36) antibody, consider these troubleshooting approaches:

Antibody Validation:

• Verify antibody specificity using dot blot analysis with peptides containing unmodified K36, K36me1, K36me2, K36me3, and K36ac .

• Ensure the antibody hasn't degraded due to improper storage; store at -20°C or -80°C and avoid repeated freeze-thaw cycles .

Sample Preparation Issues:

• Confirm proper histone extraction protocols; incomplete extraction or degradation during preparation can affect results.

• Adjust fixation conditions for immunofluorescence; 0.5% PFA fixation has been shown to work well for H3K36me1 detection in HeLa cells .

Species-Specific Considerations:

• Check if the antibody is validated for your specific species. The amino acid sequence surrounding K36 can vary between species and affect antibody recognition .

• For example, in Tetrahymena, the substitution of valine with isoleucine preceding K36 may affect antibody binding efficiency .

Application-Specific Optimization:

• For Western blots: Adjust antibody concentration, incubation time/temperature, and blocking conditions.

• For immunofluorescence: Optimize fixation method, permeabilization, antibody dilution (typical starting dilution: 1/100), and incubation time (typical: 1 hour at room temperature) .

• For ChIP assays: Optimize fixation time, sonication conditions, antibody amount, and washing stringency.

Cross-Reactivity Assessment:

• Test the antibody against recombinant histones with defined modifications to rule out cross-reactivity with other methylation states or modifications.

• Include appropriate controls in experiments, such as samples from organisms or cells lacking the enzyme responsible for H3K36 methylation (e.g., Set2 deletion mutants) .

What methodological approaches can be used to study the dynamics of H3K36 methylation during transcription elongation?

To study the dynamics of H3K36 methylation during transcription elongation, researchers can employ several methodological approaches:

Chromatin Immunoprecipitation (ChIP) Analysis:

• Perform ChIP with H3K36me1-specific antibodies followed by qPCR or sequencing to map genomic distribution .

• Design primers targeting different regions of genes (promoters, 5' regions, middle, and 3' regions) to analyze the distribution pattern along transcription units .

• Compare H3K36me1 patterns with RNA polymerase II occupancy using appropriate antibodies against different phosphorylation states of the polymerase .

Genetic Manipulation Studies:

• Utilize cells or organisms with deletions or mutations in Set2 or other H3K36 methyltransferases to assess the effects on transcription elongation .

• Employ RNA polymerase II mutants affecting elongation to study consequent changes in H3K36 methylation patterns .

Time-Course Experiments:

• Induce transcription of specific genes using appropriate stimuli and perform time-course ChIP experiments to track changes in H3K36 methylation.

• Correlate with RNA-seq data to associate methylation dynamics with transcriptional output.

Live-Cell Imaging:

• Develop fluorescent reporter systems for visualizing H3K36 methylation and transcription elongation in living cells.

• Employ FRAP (Fluorescence Recovery After Photobleaching) to study the dynamics of proteins involved in H3K36 methylation.

Nascent RNA Analysis:

• Combine H3K36me1 ChIP with nascent RNA sequencing techniques (e.g., GRO-seq, NET-seq) to directly correlate methylation with active transcription.

• Use pulse-chase approaches to track how newly synthesized RNA correlates with changes in H3K36 methylation.

Mass Spectrometry Time-Course:

• Perform quantitative mass spectrometry at different time points following transcriptional induction to measure changes in H3K36 methylation levels.

• This approach can distinguish between different methylation states (mono-, di-, and tri-) and their relative abundances during transcription.

How should researchers interpret differences in H3K36 mono-methylation patterns between species?

When interpreting differences in H3K36 mono-methylation patterns between species, researchers should consider several factors:

Evolutionary Conservation and Divergence:

• H3K36 methylation is conserved across diverse eukaryotes, suggesting fundamental importance, but the relative abundance and distribution can vary significantly .

• Consider that in some organisms like Tetrahymena thermophila, variations in the amino acid sequence surrounding K36 (isoleucine instead of valine preceding K36) may affect both antibody recognition and functional outcomes .

Technical Considerations:

• Antibody cross-reactivity and specificity may vary between species due to slight variations in histone sequences .

• Ensure that appropriate antibody validation has been performed for each species under investigation.

Functional Context:

Data Normalization:

• When comparing H3K36me1 levels between species, normalization approaches should account for differences in histone variant abundance and genomic organization.

• Consider using ratios of modified to unmodified H3 rather than absolute levels for more meaningful comparisons.

Integrated Analysis:

• Integrate data on other histone modifications to understand species-specific patterns in the context of the broader histone code.

• Consider genome size, gene density, and chromatin organization differences between species when interpreting distribution patterns.

What controls should be included when validating the specificity of Mono-methyl-HIST1H3A (K36) antibody?

Proper validation of Mono-methyl-HIST1H3A (K36) antibody specificity requires comprehensive controls:

Peptide Competition Assays:

• Pre-incubate the antibody with excess K36me1 peptide to block specific binding .

• Parallel experiments with non-specific peptides should not affect antibody binding.

Peptide Array Analysis:

• Test antibody binding against a panel of peptides containing:

  • Unmodified K36

  • K36me1, K36me2, K36me3 (different methylation states)

  • K36ac (acetylation at the same position)

  • Modifications at different lysine residues (e.g., K4me1, K9me1, K27me1)

• Use increasing peptide concentrations (e.g., 0.3-10 picomoles) to assess sensitivity and specificity thresholds .

Genetic Controls:

• Analyze samples from organisms or cells lacking the enzyme responsible for H3K36 methylation (e.g., Set2 deletion mutants) .

• These samples should show reduced or absent signal with a specific H3K36me1 antibody.

Histone Methyltransferase Assays:

• Perform in vitro methylation of recombinant H3 or nucleosomes using Set2 or other H3K36 methyltransferases .

• Compare antibody reactivity before and after methylation.

• Include assays with H3 peptides containing K36 mutations (K36A or K36R) as negative controls .

Cross-Species Validation:

• Test antibody performance across multiple species with known conservation of K36 methylation (e.g., human, chicken, yeast) .

• Account for sequence variations surrounding K36 that might affect antibody recognition.

Application-Specific Controls:

• For Western blot: Include recombinant histones with defined modifications and samples with known H3K36me1 status .

• For immunofluorescence: Include appropriate counterstains (e.g., DAPI for nuclei, α-tubulin) and perform peptide competition controls .

• For ChIP: Include input controls, IgG controls, and positive/negative control regions with known H3K36me1 status.

What emerging technologies might enhance the study of H3K36 mono-methylation in chromatin regulation?

Several emerging technologies hold promise for advancing our understanding of H3K36 mono-methylation:

Single-Cell Epigenomics:

• Single-cell ChIP-seq and CUT&Tag methods will enable examination of H3K36me1 heterogeneity within cell populations.

• These approaches can reveal cell-type-specific patterns and dynamics of H3K36 methylation that are masked in bulk analysis.

Genome Editing for Histone Mutations:

• CRISPR-Cas9 systems for precise editing of histone genes to create K36 mutations or methylation-mimicking substitutions.

• Development of inducible systems to temporally control H3K36 methylation status.

Live-Cell Epigenetic Imaging:

• Development of specific histone modification sensors for real-time visualization of H3K36me1 dynamics in living cells.

• Multiplexed imaging systems to simultaneously track multiple histone modifications alongside transcriptional activity.

Proximity Ligation Technologies:

• Techniques like ChIA-PET and HiChIP adapted specifically for H3K36me1 to understand three-dimensional chromatin organization in relation to this modification.

• These approaches can reveal how H3K36me1 influences long-range chromatin interactions and nuclear compartmentalization.

Mass Spectrometry Innovations:

• Improvements in top-down proteomics to identify combinatorial patterns of histone modifications co-occurring with H3K36me1 on individual histone molecules.

• Development of targeted mass spectrometry approaches for more sensitive quantification of specific histone modifications.

Long-Read Sequencing Applications:

• Adaptation of long-read sequencing technologies (Nanopore, PacBio) for direct detection of histone modifications.

• These approaches could potentially reveal long-range patterns and relationships between H3K36me1 and other chromatin features.

Cryo-EM and Structural Studies:

• High-resolution structural studies of nucleosomes containing H3K36me1 to understand how this modification affects nucleosome structure and dynamics.

• Investigation of reader protein complexes that specifically recognize H3K36me1 to elucidate downstream effector mechanisms.