Mono-methyl-Histone H3.1 (K36) Recombinant Monoclonal Antibody

What is Mono-methyl-Histone H3.1 (K36) and why is it significant in epigenetic research?

Mono-methyl-Histone H3.1 (K36) refers to the histone H3.1 protein with a single methyl group attached to the lysine residue at position 36. Histone H3.1 is one of the five main histone proteins involved in chromatin structure in eukaryotic cells. It features a main globular domain and a long N-terminal tail that protrudes from the nucleosome core. This N-terminal tail can undergo various epigenetic modifications, including methylation at specific lysine residues like K36. These modifications influence critical cellular processes by altering chromatin structure and accessibility . Mono-methylation at K36 specifically functions in transcriptional repression and chromatin compaction, with additional roles in processes such as alternative splicing, DNA repair, cellular identity maintenance, and epigenetic memory. This specific modification represents a crucial epigenetic mark that helps regulate gene expression patterns and chromatin architecture .

What are the recommended applications and dilutions for Mono-methyl-Histone H3.1 (K36) antibodies?

The Mono-methyl-Histone H3.1 (K36) antibodies can be utilized in multiple experimental applications with specific recommended dilution ranges for optimal results:

These dilution ranges should be optimized based on sample type, antibody lot, and specific experimental conditions . For Western blot applications, researchers should first validate the antibody specificity using positive and negative controls. For immunofluorescence and immunocytochemistry applications, fixation method can significantly impact antibody performance, with paraformaldehyde fixation typically yielding optimal results for nuclear epitopes.

What storage and handling conditions are recommended for these antibodies?

Proper storage and handling of Mono-methyl-Histone H3.1 (K36) antibodies are essential for maintaining their specificity and activity:

• Storage temperature: Store at -20°C for up to 12 months

• For longer-term storage, -80°C is recommended to prevent degradation

• Avoid repeated freeze-thaw cycles by aliquoting the antibody upon receipt

• The antibody is typically supplied in a storage buffer containing:

  • Aqueous buffered solution with 1xTBS (pH 7.4)

  • 1% BSA as a stabilizer

  • 40% Glycerol as a cryoprotectant

  • 0.05% Sodium Azide as a preservative

For optimal performance, thaw antibody aliquots on ice before use and keep cold during handling. Return to -20°C promptly after use to preserve activity and specificity for future experiments.

How does mono-methylation at H3K36 functionally differ from di- and tri-methylation states?

The different methylation states at H3K36 (mono-, di-, and tri-methylation) have distinct functional consequences and are regulated by different methyltransferases. Mono-methylation at K36 primarily functions in transcriptional repression and chromatin compaction, while tri-methylation is generally associated with actively transcribed regions .

Research has shown that increasing levels of methylation at K36 negatively impact the interaction of certain proteins with histone H3.1. For example, the TONSOKU (TSK) protein's TPR domain shows decreased binding affinity to H3.1 as methylation levels increase at K36 . This differential binding behavior suggests that mono-methylation serves as a distinct functional state that is recognized by specific reader proteins.

The structural basis for this discrimination has been elucidated through crystallography, revealing that the TPR domain of TSK forms a deep pocket that accommodates the side chain of H3.1K36. The ε-amine group of K36 is positioned in close proximity to the carboxyl group of Asp54, creating a specific interaction that is sensitive to methylation state . This provides a molecular mechanism for how reader proteins can distinguish between different methylation states at the same residue.

What is the role of H3K36 mono-methylation in DNA repair and genomic stability?

H3K36 mono-methylation plays a critical role in DNA repair pathways and maintaining genomic stability. Recent research has revealed an H3.1-specific function during DNA replication and repair that is regulated by the methylation status of K36 .

The TONSOKU (TSK) protein, which is involved in DNA repair processes, preferentially binds to unmethylated or lowly methylated H3.1 at K36. Crystal structure analysis has shown that TPR TSK forms a pocket that accommodates the side chain of H3.1K36, where its ε-amine is in close proximity to the carboxyl group of Asp54. This pocket has a polarity that makes it non-conducive for binding hydrophobic moieties such as methyl groups, explaining the decreased binding affinity when K36 is methylated .

This molecular mechanism suggests that newly synthesized H3.1 (which would be unmethylated at K36) can recruit TSK to sites of DNA damage or replication stress. As methylation at K36 increases during or after DNA repair, TSK binding decreases, potentially signaling the completion of repair processes. This represents a common strategy used in multicellular eukaryotes for regulating DNA repair through histone variant-specific modifications .

How does H3K36 mono-methylation interact with histone deacetylation pathways?

H3K36 methylation plays a crucial role in recruiting histone deacetylase complexes to maintain appropriate chromatin structure during transcription elongation. Research has revealed a trans-histone pathway where H3K36 methylation leads to deacetylation of histones H3 and H4 .

This process occurs through the specific recognition of H3K36 methylated histones by the chromodomain (CHD) of Eaf3 and the plant homeobox domain (PHD) of Rco1, which are two subunits of the Rpd3S histone deacetylase complex. After recognition, the Rpd3S complex deacetylates histones H3 and H4 within the open reading frame (ORF) regions .

The functional consequence of this interaction is significant: it prevents spurious intragenic transcription that could otherwise initiate from cryptic promoters within gene bodies. This represents a key regulatory mechanism ensuring transcriptional fidelity across the genome. Interestingly, while the recognition of H3K36 methylation is critical for this process, studies have shown that global H3K36 trimethylation levels are not affected by certain mutations in histone H4 (such as H4 V43A), suggesting that there are multiple regulatory inputs controlling this pathway .

What validation methods should researchers employ to confirm antibody specificity for mono-methyl H3K36?

Rigorous validation of antibody specificity is crucial when working with histone modification-specific antibodies like Mono-methyl-Histone H3.1 (K36). Recommended validation approaches include:

• Peptide Competition Assays: Pre-incubate the antibody with increasing concentrations of:

  • Mono-methylated H3K36 peptide (should block signal)

  • Unmethylated H3K36 peptide (should not block signal)

  • Di- or tri-methylated H3K36 peptide (should not block signal)

• Positive/Negative Controls:

  • Use cell lines with known H3K36me1 levels

  • Compare with samples treated with methyltransferase inhibitors

  • Test in knockout/knockdown models of H3K36 methyltransferases

• Peptide Array Analysis: Test antibody against a panel of modified histone peptides to assess cross-reactivity with other modifications .

• Orthogonal Method Validation: Compare results with other techniques that can detect H3K36 methylation, such as mass spectrometry.

A well-validated antibody should demonstrate high specificity for mono-methylated H3K36 with minimal cross-reactivity to unmethylated H3K36 or other methylation states (di- or tri-methylation). Researchers should document these validation steps in publications to ensure reproducibility.

What factors should researchers consider when designing experiments with H3K36me1 antibodies?

When designing experiments with Mono-methyl-Histone H3.1 (K36) antibodies, researchers should consider several critical factors:

• Antibody Clone Selection: Different clones may have varying specificities and performance characteristics. For example, clone 6G6 and 1F4 are mentioned in the search results and may perform differently based on the application .

• Cell Type Considerations: The abundance and distribution of H3K36me1 can vary significantly between cell types and tissues. Consider using positive control cell lines with known H3K36me1 levels.

• Chromatin Context: The accessibility of the H3K36me1 epitope can be affected by chromatin compaction and neighboring modifications. Optimize fixation and permeabilization protocols accordingly.

• Cross-reactivity Assessment: Test for potential cross-reactivity with other histone modifications, particularly di- and tri-methylated H3K36, as well as methylation at other lysine residues like K4, K9, and K27 .

• Technical Controls:

  • Include isotype controls

  • Use competitive peptide blocking

  • Include samples with enzymatically removed modifications

• Experimental Timing: Consider cell cycle phase, as histone modification patterns can change throughout the cell cycle, particularly for replication-dependent variants like H3.1.

How can researchers troubleshoot common issues when using this antibody in different applications?

When troubleshooting experiments with Mono-methyl-Histone H3.1 (K36) antibodies, consider the following application-specific strategies:

For Western Blot issues:

• Weak or no signal: Increase antibody concentration (start with 1:500 and adjust), optimize protein extraction methods to preserve modifications, or use enhanced chemiluminescence detection systems

• High background: Increase blocking time/concentration, reduce primary antibody concentration, or add 0.1% Tween-20 to washing buffers

• Multiple bands: Test specificity with peptide competition assays, optimize extraction to minimize histone degradation

For Immunofluorescence issues:

• Nuclear exclusion: Optimize fixation and permeabilization methods to maintain nuclear integrity while allowing antibody access

• Weak signal: Try antigen retrieval methods, increase antibody concentration (starting at 1:50), or extend incubation time

• Non-specific staining: Increase blocking time with 5% BSA or normal serum, reduce antibody concentration, or perform additional washes

For ChIP applications:

• Low enrichment: Optimize crosslinking conditions, increase chromatin sonication efficiency, or adjust antibody-to-chromatin ratio

• High background: Pre-clear chromatin with protein A/G beads, use more stringent washing conditions, or add competitor DNA (e.g., salmon sperm DNA)

What are the structural determinants of antibody specificity for H3K36me1 versus other methylation states?

The structural basis for antibody specificity toward mono-methylated H3K36 involves precise molecular recognition of both the lysine side chain and its specific methylation state. Multiple factors contribute to this specificity:

• Recognition Pocket Architecture: High-specificity antibodies possess a binding pocket that can accommodate the mono-methyl group but sterically excludes di- or tri-methyl groups. The crystal structure of proteins that interact with H3K36 provides insights into how this discrimination might occur. For example, the TPR domain of TONSOKU forms a pocket where the ε-amine of K36 interacts with Asp54 . Similar structural features likely exist in antibodies specific for H3K36me1.

• Hydrogen Bonding Network: Mono-methylation leaves the lysine nitrogen with the capability to form hydrogen bonds, unlike tri-methylation which eliminates this potential. Antibodies that recognize H3K36me1 often form critical hydrogen bonds with the remaining hydrogen of the mono-methylated lysine.

• Surrounding Amino Acid Context: The antibody recognition extends beyond just the modified lysine to include surrounding amino acids. The sequence context around K36 (particularly A31-R40) creates a specific binding interface that contributes to specificity .

• Cation-π Interactions: Aromatic residues in the antibody binding pocket can form favorable interactions with the positively charged methylated lysine, with the strength of these interactions varying based on methylation state.

Understanding these structural determinants helps in designing validation experiments and interpreting cross-reactivity patterns when working with these antibodies.

What are the latest research findings on the biological significance of H3K36 mono-methylation?

Recent research has revealed several important roles for H3K36 mono-methylation in cellular processes:

• Transcriptional Regulation: H3K36 mono-methylation has been identified as a key regulator in transcriptional repression and chromatin compaction, helping to maintain proper gene expression patterns .

• DNA Repair Pathway Regulation: Studies have uncovered that H3K36 mono-methylation status affects the binding of DNA repair proteins such as TONSOKU (TSK). The TPR domain of TSK shows decreased binding affinity to H3.1 as methylation levels increase at K36, suggesting that mono-methylation serves as a regulatory mechanism for recruiting repair factors to damaged DNA .

• Cellular Identity Maintenance: H3K36 mono-methylation contributes to maintaining cellular identity by stabilizing expression patterns of cell-type-specific genes, functioning as part of the epigenetic memory system .

• Alternative Splicing Regulation: This modification has been implicated in regulating alternative splicing processes, potentially by recruiting or excluding splicing factors from chromatin regions .

• Trans-histone Regulatory Pathways: Research has revealed that H3K36 methylation participates in trans-histone pathways, where it influences the deacetylation of histones H3 and H4 via the Rpd3S histone deacetylase complex, preventing spurious intragenic transcription .

These findings collectively highlight H3K36 mono-methylation as a multifunctional epigenetic mark with roles extending beyond basic chromatin structure to include complex regulatory mechanisms in gene expression, DNA repair, and cellular identity.

How is H3K36 mono-methylation being studied in the context of disease models?

H3K36 mono-methylation has emerged as an important epigenetic mark with implications in various disease contexts. While the search results don't provide comprehensive information on disease models, they do mention that H3K36 mono-methylation has "implications in disease" . Based on current scientific understanding, researchers are investigating this modification in several disease contexts:

• Cancer Biology: Altered patterns of H3K36 methylation have been observed in various cancer types. Researchers are using H3K36me1-specific antibodies to map genome-wide distribution changes in cancer cells compared to normal tissues.

• Neurodevelopmental Disorders: Given the role of H3K36 methylation in transcriptional regulation and cellular identity, researchers are examining its potential dysregulation in neurodevelopmental conditions.

• DNA Repair Deficiency Syndromes: Since H3K36 mono-methylation affects interactions with DNA repair proteins like TONSOKU , researchers are investigating whether aberrant H3K36 methylation contributes to genomic instability in DNA repair-deficient conditions.

• Stem Cell Models: The role of H3K36 mono-methylation in maintaining cellular identity makes it relevant for stem cell research and regenerative medicine applications.

Methodologically, these studies often combine ChIP-seq using H3K36me1-specific antibodies with transcriptome analysis and functional assays to establish causative relationships between altered methylation patterns and disease phenotypes.

What emerging technologies are enhancing the study of site-specific histone modifications like H3K36me1?

Several cutting-edge technologies are revolutionizing how researchers study site-specific histone modifications like H3K36 mono-methylation:

• CUT&RUN and CUT&Tag: These antibody-directed genomic mapping techniques offer advantages over traditional ChIP-seq, including lower input requirements, reduced background, and improved signal-to-noise ratio. They are particularly valuable for mapping H3K36me1 distribution in rare cell populations or clinical samples.

• Single-Cell Epigenomics: Techniques like single-cell CUT&Tag and scATAC-seq combined with antibody-based approaches allow researchers to examine H3K36me1 distribution at the single-cell level, revealing cell-to-cell variability in modification patterns.

• Mass Spectrometry Advances: Quantitative MS approaches can now detect and quantify specific histone modifications with high sensitivity, allowing researchers to measure absolute levels of H3K36me1 and other modifications simultaneously.

• Proximity Ligation Assays: These techniques enable the visualization and quantification of specific histone modifications in situ, providing spatial information about H3K36me1 distribution within the nucleus.

• CRISPR-Based Epigenome Editing: Targeted modification of H3K36 methylation status at specific genomic loci is now possible using CRISPR-dCas9 fused to methyltransferases or demethylases, allowing for cause-effect studies.

• Recombinant Antibody Technology: The development of recombinant monoclonal antibodies against H3K36me1 has improved specificity and reproducibility compared to traditional polyclonal antibodies . These antibodies are produced through cloning of genes encoding the antibody and expression in mammalian cell systems, followed by purification using affinity chromatography.

These technological advances are enabling researchers to gain unprecedented insights into the distribution, dynamics, and functional significance of H3K36 mono-methylation across diverse biological contexts.

What are the key considerations when selecting and validating a Mono-methyl-Histone H3.1 (K36) antibody?

When selecting and validating a Mono-methyl-Histone H3.1 (K36) antibody for research applications, several critical factors should be considered:

• Specificity: The antibody should specifically recognize mono-methylated K36 on histone H3.1 with minimal cross-reactivity to unmethylated H3K36 or other methylation states (di-, tri-methylation). Validation through peptide competition assays and testing against methylation site mutants is essential .

• Application Compatibility: Different antibody clones may perform optimally in specific applications. Consider whether the antibody has been validated for your intended application (WB, IF, ICC, ChIP, etc.) and at what recommended dilutions .

• Host Species and Clonality: Monoclonal antibodies like clones 6G6 and 1F4 offer consistent lot-to-lot reproducibility and high specificity. Consider the host species (rabbit in most cases for these antibodies) to avoid cross-reactivity in multi-labeling experiments .

• Production Method: Recombinant monoclonal antibodies produced in mammalian expression systems often offer superior consistency and specificity compared to hybridoma-derived antibodies. These are produced by cloning both heavy and light chain genes into expression vectors, followed by expression in mammalian cells and purification via affinity chromatography .

• Validation Documentation: Review the manufacturer's validation data, including Western blot images, peptide array results, and immunofluorescence images to assess performance characteristics before selection.

By carefully evaluating these factors, researchers can select antibodies that will provide reliable, reproducible results in their specific experimental context, enabling accurate characterization of H3K36 mono-methylation patterns and functions.

How does the study of H3K36 mono-methylation contribute to our broader understanding of epigenetic regulation?

The study of H3K36 mono-methylation has significantly enhanced our understanding of epigenetic regulation in several fundamental ways:

• Transcriptional Regulation Mechanisms: H3K36 mono-methylation contributes to our understanding of how histone modifications regulate gene expression through its role in transcriptional repression and chromatin compaction . This modification helps establish the "histone code" that determines which genes are expressed in specific cellular contexts.

• Histone Modification Crosstalk: Research on H3K36me1 has revealed important insights into how different histone modifications interact in trans-histone regulatory pathways. For example, H3K36 methylation leads to histone deacetylation through the recruitment of the Rpd3S complex , demonstrating how modifications on one histone can influence modifications on other histones.

• Chromatin Dynamics During DNA Replication and Repair: Studies of H3K36me1 have illuminated how histone modifications are regulated during DNA replication and repair processes. The interaction between H3K36 methylation status and DNA repair proteins like TONSOKU provides a molecular mechanism for how chromatin modifications can regulate genome maintenance .

• Variant-Specific Functions: Research has revealed that histone variants (like H3.1 versus H3.3) can have distinct functions through their specific modification patterns. H3.1-specific functions during replication and DNA repair highlight how the cell uses variant-specific modifications as regulatory mechanisms .

• Epigenetic Inheritance and Memory: H3K36 mono-methylation contributes to cellular identity and epigenetic memory, providing insights into how cells maintain their differentiation status through cell divisions .