Mono-methyl-Histone H2B type 2-E (R79) Recombinant Monoclonal Antibody

What is Mono-methyl-Histone H2B type 2-E (R79) and why is it significant in epigenetic research?

Mono-methyl-Histone H2B type 2-E (R79) represents a specific post-translational modification where the arginine residue at position 79 of histone H2B type 2-E undergoes mono-methylation. Histone H2B serves as a core component of nucleosomes, which wrap and compact DNA into chromatin, thereby regulating accessibility to the cellular machinery requiring DNA as a template. This specific modification contributes to the complex "histone code" that regulates transcription, DNA repair, replication, and chromosomal stability . The mono-methylation at R79 is part of the intricate regulatory system that determines chromatin structure and gene expression patterns.

What are the technical specifications of Mono-methyl-Histone H2B type 2-E (R79) Recombinant Monoclonal Antibody?

The following table outlines the key specifications of the Mono-methyl-Histone H2B type 2-E (R79) Recombinant Monoclonal Antibody:

This recombinant monoclonal antibody is produced in HEK293F cells, ensuring consistent lot-to-lot reproducibility compared to traditional hybridoma-derived antibodies .

How can Mono-methyl-Histone H2B (R79) antibody be incorporated into chromatin immunoprecipitation protocols?

When incorporating this antibody into chromatin immunoprecipitation (ChIP) protocols, researchers should consider the following methodological approach:

• Crosslinking preparation: Begin with a formaldehyde crosslinking step (typically 1% for 10 minutes at room temperature) to preserve protein-DNA interactions.

• Chromatin preparation: Sonicate chromatin to fragments of approximately 200-500 bp, which is optimal for histone modification studies.

• Antibody specificity verification: Prior to full-scale experiments, verify antibody specificity using peptide competition assays or knockout/knockdown controls to ensure specificity for mono-methyl-H2B (R79).

• Antibody concentration optimization: Typically, 2-5 μg of antibody per ChIP reaction is recommended, but this should be empirically determined for each experimental system.

• Spike-in controls: Include DNA-barcoded PTM-defined spike-in nucleosome standards to provide an in situ metric of target enrichment, similar to the Kub1Stat spike-in controls used for other histone modifications .

• Sequential ChIP considerations: For studies examining co-occurrence with other histone modifications, sequential ChIP may be performed with appropriate controls for antibody removal between rounds.

• Data analysis: Align reads to the appropriate genome (e.g., T2T genome for human studies) and analyze enrichment patterns in relation to genomic features such as promoters, enhancers, and gene bodies .

What experimental approaches can reveal the functional relationship between H2B methylation and ubiquitination?

To investigate the functional relationship between H2B methylation and ubiquitination, researchers can employ several sophisticated approaches:

• Sequential ChIP-seq analysis: Perform ChIP-seq first with anti-H2BK120ub1 antibody followed by anti-mono-methyl-H2B (R79) to identify genomic regions where both modifications co-occur.

• Enzyme activity assays: Similar to the experiments described for COMPASS activity, prepare reaction mixtures containing purified COMPASS components, nucleosomes with H2B-Ub, and SAM (S-adenosyl methionine) as a methyl donor, then measure the effect of H2B mono-methylation at R79 on enzyme kinetics .

• Mutation studies: Generate site-specific mutations at R79 (e.g., R79K or R79A) in H2B and analyze the effects on H2B ubiquitination patterns using western blotting or mass spectrometry.

• Temporal dynamics analysis: Use time-course experiments with ChIP-seq or biochemical assays to determine whether H2B ubiquitination precedes or follows R79 methylation during transcriptional activation.

• Structural biology approaches: Similar to studies with COMPASS, use cryo-EM to determine whether H2B-R79 methylation affects the structural conformation of nucleosomes and their interaction with ubiquitination machinery .

These approaches can help determine whether these modifications function in a cooperative, antagonistic, or independent manner, contributing to our understanding of the "trans-histone crosstalk" phenomenon .

How should researchers validate the specificity of Mono-methyl-Histone H2B (R79) antibody in multiplex histone modification studies?

Validating antibody specificity is crucial, especially in multiplex studies examining multiple histone modifications simultaneously. Researchers should employ the following comprehensive approach:

• Peptide array screening: Test the antibody against synthetic peptides containing mono-methyl-R79 as well as peptides with other methylation states (di/tri-methyl) and other arginine methylation sites in histones to confirm site-specificity.

• Multiplexed nucleosome panel testing: Use a dCypher Luminex assay platform where biotinylated nucleosomes or peptides are bound to streptavidin-coated, uniquely identifiable xMAP beads. The antibody should be tested at multiple dilutions (e.g., 1:250, 1:1000, 1:4000) against various modified nucleosomes to determine percent specificity .

• Competition assays: Perform antibody binding in the presence of excess free peptide containing the mono-methyl-R79 modification to demonstrate binding is specific to the modification of interest.

• Western blot with controls: Include positive controls (cells known to have the modification) and negative controls (cells where the modification has been enzymatically removed or prevented).

• Knockout/knockdown validation: Use cells where the enzyme responsible for R79 methylation has been depleted to verify reduced signal.

• Cross-reactivity assessment: Generate a cross-reactivity matrix testing against all major histone modifications, particularly those that commonly co-occur with H2B modifications.

The specificity data should be quantified using median fluorescence intensity (MFI) normalized to on-target signal, with >80% specificity generally considered acceptable for chromatin studies .

What bioinformatic approaches are most effective for analyzing the genomic distribution of Mono-methyl-Histone H2B (R79) in relation to gene expression?

The following bioinformatic approaches are recommended for analyzing the relationship between Mono-methyl-Histone H2B (R79) distribution and gene expression:

• Peak calling and annotation: Use specialized peak callers optimized for histone modifications (e.g., MACS2 with broad peak settings) followed by annotation to genomic features using tools like ChIPseeker or HOMER.

• Correlation with transcriptome data: Generate heatmaps sorting genes by expression levels (from RNA-seq data) and visualize the corresponding distribution of H2B-R79me1, similar to analyses done for other histone modifications .

• Integrative genomic viewer (IGV) visualization: Compare genomic enrichment patterns of H2B-R79me1 with other histone marks such as H3K27me1/2/3, H3K36me3, and H2BK120ub1 to identify potential relationships .

• Metagene analysis: Generate aggregate plots showing the average enrichment pattern around transcription start sites (TSS), gene bodies, and transcription end sites (TES) for genes grouped by expression levels.

• Co-occurrence analysis: Calculate the Jaccard index or other similarity metrics between H2B-R79me1 peaks and other histone modifications to quantify the degree of overlap.

• Machine learning approaches: Apply supervised learning algorithms to predict gene expression levels based on H2B-R79me1 and other histone modification patterns.

• Motif analysis: Identify DNA sequence motifs enriched in regions with H2B-R79me1 to identify potential transcription factor associations.

This analytical framework will help determine whether H2B-R79me1 is associated with active transcription (like H3K4me3, H3K36me3, and H2BK120ub1) or repressed genes (like H3K27me3 and H2AK119ub1) .

How does Mono-methyl-Histone H2B (R79) contribute to the trans-histone crosstalk mechanism?

Trans-histone crosstalk refers to the phenomenon where modifications on one histone influence modifications on another histone. While the search results don't specifically detail H2B-R79me1's role in this process, we can extrapolate from related histone modification crosstalk mechanisms:

• Potential effector recruitment: H2B-R79me1 may serve as a binding site for specific "reader" proteins that subsequently recruit or activate enzymes responsible for modifying other histones.

• Structural implications: Similar to how H2B ubiquitination affects nucleosome structure and subsequently COMPASS activity on H3K4 , H2B-R79me1 might induce conformational changes in the nucleosome that affect accessibility of other histone residues to modifying enzymes.

• Enzymatic regulation: The methylation at R79 could potentially regulate the activity of histone-modifying enzymes directly, similar to how H2B ubiquitination stimulates methyltransferase activity without necessarily increasing enzyme binding affinity .

• Sequential modification patterns: H2B-R79me1 might be part of a sequential modification pattern that includes H2B ubiquitination and subsequent H3 methylation, contributing to the established crosstalk between H2B and H3 .

• Competitive modification: R79 methylation might compete with other potential modifications at or near this residue, indirectly affecting histone crosstalk pathways.

Further research using techniques such as sequential ChIP, mass spectrometry, and structural studies is needed to fully elucidate the specific role of H2B-R79me1 in trans-histone crosstalk mechanisms.

What methodological approaches can distinguish the specific effects of H2B-R79 methylation from other concurrent histone modifications?

Distinguishing the specific effects of H2B-R79 methylation from other concurrent modifications requires sophisticated experimental approaches:

• Site-specific histone mutants: Generate histone H2B with R79K or R79A mutations that prevent methylation specifically at this site while preserving the ability to receive other modifications.

• Reconstituted nucleosomes with defined modifications: Use semi-synthetic or enzymatically modified recombinant histones to create nucleosomes with specific combinations of modifications for in vitro activity assays .

• Methyltransferase inhibitors/knockdowns: Selectively inhibit or deplete the arginine methyltransferase responsible for R79 methylation while monitoring the effects on other histone modifications.

• Mass spectrometry-based proteomics: Perform quantitative MS analysis to correlate H2B-R79me1 with other modifications at the single-nucleosome level, identifying statistically significant co-occurrences or mutual exclusions.

• Single-molecule approaches: Utilize single-molecule imaging techniques to track the temporal order of modification deposition and removal in living cells.

• Crosslinking mass spectrometry: Apply this technique to identify proteins that specifically interact with H2B-R79me1 but not with unmodified H2B or other modified forms .

• Reader domain binding assays: Test binding of various chromatin-associated protein domains to peptides or nucleosomes containing H2B-R79me1 alone or in combination with other modifications.

These approaches can help isolate the specific consequences of H2B-R79 methylation in the complex network of histone modifications.

What are the common technical challenges when using Mono-methyl-Histone H2B (R79) antibody in western blotting and how can they be overcome?

Researchers commonly encounter several technical challenges when using histone modification-specific antibodies in western blotting:

• Cross-reactivity issues:

  • Challenge: The antibody may recognize similar methylation sites on other histones.

  • Solution: Include appropriate controls such as peptide competition assays and use recombinant histone standards with defined modifications as positive controls .

• Signal intensity problems:

  • Challenge: Weak signal due to low abundance of the R79 methylation mark.

  • Solution: Enrich for histones using acid extraction protocols, optimize antibody concentration (try dilutions from 1:250 to 1:4000), and extend primary antibody incubation to overnight at 4°C .

• High background:

  • Challenge: Non-specific binding resulting in high background.

  • Solution: Increase blocking time/concentration (5% BSA often works better than milk for phospho-specific antibodies), add 0.1% Tween-20 to wash buffers, and consider using specialized low-background detection systems.

• Inconsistent results:

  • Challenge: Variation between experiments.

  • Solution: Standardize histone extraction protocols, use internal loading controls, and consider quantitative western blotting with standard curves.

• Epitope masking:

  • Challenge: Nearby modifications might mask the R79 methylation site.

  • Solution: Test different sample preparation methods, including various denaturing conditions and the use of phosphatase or deubiquitinase treatments if appropriate.

• Protein transfer issues:

  • Challenge: Poor transfer of histones due to their small size and basic nature.

  • Solution: Use PVDF membranes (rather than nitrocellulose), adjust transfer conditions (20% methanol, longer transfer times), and consider specialized transfer buffers for basic proteins.

Optimizing these parameters systematically will help achieve consistent and specific detection of Mono-methyl-Histone H2B (R79).

How can researchers optimize immunoprecipitation efficiency when studying low-abundance H2B-R79 methylation?

Optimizing immunoprecipitation (IP) efficiency for low-abundance histone modifications requires careful attention to several key parameters:

• Starting material optimization:

  • Increase the amount of starting chromatin (10-20 million cells may be necessary for low-abundance modifications).

  • Consider using cell synchronization to enrich for cell cycle phases where the modification may be more prevalent.

• Crosslinking parameters:

  • Optimize crosslinking conditions (0.1-1% formaldehyde for 5-15 minutes) to preserve protein-DNA interactions without over-crosslinking.

  • Consider dual crosslinking with DSG (disuccinimidyl glutarate) before formaldehyde for protein-protein interactions.

• Antibody strategies:

  • Use higher antibody concentrations (5-10 μg per IP) for low-abundance marks.

  • Consider a sequential IP approach where the first IP enriches for H2B, followed by a second IP with the R79me1-specific antibody.

  • Extend antibody incubation time to overnight at 4°C with gentle rotation .

• Beads and washing optimization:

  • Compare different types of beads (Protein A/G, magnetic vs. agarose).

  • Test pre-clearing the chromatin with beads alone to reduce non-specific binding.

  • Optimize wash stringency by adjusting salt concentrations in wash buffers.

• Elution and recovery techniques:

  • Compare different elution methods (SDS, competitive peptide elution, acid elution).

  • For sequential ChIP, ensure complete elution from the first IP before proceeding.

• Signal amplification methods:

  • Consider using CUT&RUN or CUT&Tag approaches which can provide improved signal-to-noise for low-abundance modifications .

  • Implement PCR optimization to amplify recovered DNA without introducing bias.

• Controls and spike-ins:

  • Include spike-in controls like Kub1Stat (for ubiquitin studies) or similar controls for methylation to normalize for IP efficiency between samples .

  • Always run parallel IPs with IgG and other control antibodies to assess background levels.

By systematically optimizing these parameters, researchers can significantly improve the detection of low-abundance H2B-R79 methylation in their experimental systems.