Mono-methyl-HIST1H2BC (K23) Antibody

What is Mono-methyl-HIST1H2BC (K23) Antibody and what does it detect?

The Mono-methyl-HIST1H2BC (K23) Antibody (PACO65045) is a rabbit polyclonal antibody that specifically recognizes the mono-methylated form of histone H2B at lysine 23 in human samples. This antibody detects a specific post-translational modification that plays a critical role in epigenetic regulation . The antibody targets a synthesized peptide derived from Human Histone H2B type 1-C/E/F/G/I protein, specifically amino acids 17-28 . This modification is part of the histone code that regulates chromatin structure and gene expression.

What applications is this antibody validated for?

This antibody has been validated for multiple experimental applications with specific recommended protocols:

The antibody demonstrates high specificity for human samples, making it particularly valuable for research on human cell lines and tissues . When designing experiments, researchers should consider the polyclonal nature of this antibody, which may provide broader epitope recognition but potentially more batch-to-batch variation compared to monoclonal alternatives.

How should this antibody be stored and handled?

For optimal performance and longevity, the antibody should be stored at -20°C or -80°C upon receipt. Repeated freeze-thaw cycles should be avoided to prevent degradation of antibody activity . The antibody is supplied in liquid form in a preservative buffer containing 0.03% Proclin 300, 50% Glycerol, and 0.01M PBS (pH 7.4) . This formulation helps maintain stability during storage. For long-term experiments, aliquoting the antibody into smaller volumes before freezing is recommended to minimize freeze-thaw cycles.

What controls should be included when working with this antibody?

When designing experiments using the Mono-methyl-HIST1H2BC (K23) Antibody, several controls are essential for validating results:

• Positive Control: Cell lines or tissues known to express mono-methylated H2B K23

• Negative Control: Samples where the modification is absent (e.g., cells treated with specific demethylase enzymes)

• Peptide Competition: Pre-incubation of antibody with the immunizing peptide to confirm specificity

• Secondary Antibody Control: Omission of primary antibody to assess nonspecific binding

• Loading Control: For western blot applications, include total H2B antibody on parallel samples

These controls help distinguish between true signal and background, especially important given the subtle nature of histone modifications and their dynamic regulation in different cellular contexts.

What is the recommended protocol for immunofluorescence studies?

For optimal immunofluorescence results with this antibody, the following protocol is recommended:

• Fix cells with 4% paraformaldehyde (15 minutes at room temperature)

• Permeabilize with 0.2% Triton X-100 (10 minutes)

• Block with 5% BSA in PBS (1 hour)

• Incubate with primary antibody at 1:10-1:100 dilution (overnight at 4°C)

• Wash 3x with PBS (5 minutes each)

• Incubate with fluorophore-conjugated secondary antibody (1 hour at room temperature)

• Counterstain nuclei with DAPI

• Mount and image using confocal or fluorescence microscopy

When imaging, the mono-methylated H2B K23 signal should appear predominantly nuclear with potential variations in intensity across different nuclear regions, reflecting the distribution of this modification within chromatin domains.

How can Western blot conditions be optimized for this antibody?

While specific Western blot conditions for this antibody aren't detailed in the source materials, protocols for similar histone modification antibodies can be adapted:

• Use SDS-PAGE gels with higher percentage (15-18%) to effectively resolve histone proteins (~17 kDa)

• Transfer to nitrocellulose or PVDF membranes using standard protocols

• Block with 5% BSA in TBST (not milk, which contains proteins that may cross-react)

• Incubate with primary antibody (starting at 1:1000 dilution)

• Use HRP-conjugated secondary antibodies for detection

• Consider enhanced chemiluminescence (ECL) for detection

Researchers should note that histone H2B typically appears at approximately 17 kDa on Western blots, slightly higher than its predicted molecular weight of 14 kDa due to post-translational modifications . Acid extraction of histones is often recommended for cleaner results with histone antibodies.

How does mono-methylation at lysine 23 of histone H2B relate to epigenetic regulation?

Mono-methylation of histone H2B at lysine 23 is part of the complex histone code that regulates chromatin accessibility and gene expression. This modification plays a crucial role in gene regulation and chromatin structure , often working in concert with other histone modifications to establish specific chromatin states. Current research suggests this modification may be involved in:

• Transcriptional regulation of specific gene sets

• DNA damage response pathways

• Cell cycle progression

• Establishment of specialized chromatin domains

Understanding the context-specific roles of this modification requires integration of data from multiple approaches, including ChIP-seq, mass spectrometry, and functional genomics.

What methodological considerations are important for quantifying histone H2B mono-methylation levels?

Accurate quantification of H2B K23 mono-methylation requires careful methodological considerations:

• Extraction Protocol: Use acid extraction (0.2N HCl) to efficiently isolate histones while preserving modifications

• Normalization Strategy: Normalize to total H2B levels to account for variations in histone content

• Technical Replicates: Include at least three technical replicates per biological sample

• Standard Curves: For ELISA applications, establish standard curves using synthetic modified peptides

• Mass Spectrometry Validation: Consider orthogonal validation using targeted mass spectrometry

When comparing methylation levels across different experimental conditions, consistent sample preparation and analysis methods are essential to minimize technical variability that could mask biological differences.

How can ChIP-seq experiments be optimized using this antibody?

For researchers conducting ChIP-seq experiments to map genome-wide distribution of H2B K23 mono-methylation, several optimization steps are recommended:

• Chromatin Preparation: Optimize sonication conditions to achieve fragments of 200-500 bp

• Antibody Amount: Titrate antibody amounts (2-10 μg per ChIP reaction) to determine optimal signal-to-noise ratio

• Input Controls: Include input DNA controls and IgG controls for each experimental condition

• Sequential ChIP: Consider sequential ChIP (re-ChIP) to identify genomic regions with co-occurrence of multiple modifications

• Library Preparation: Use library preparation methods that accommodate limited DNA amounts typical of histone modification ChIPs

During bioinformatic analysis, researchers should look for enrichment patterns in specific genomic features (promoters, enhancers, gene bodies) and correlate these patterns with transcriptional states and other epigenetic marks.

What are common issues and solutions when working with histone modification antibodies?

Researchers working with histone modification antibodies like Mono-methyl-HIST1H2BC (K23) may encounter several challenges:

These issues highlight the importance of thorough validation and optimization when working with histone modification antibodies in different experimental contexts.

How can conflicting data be resolved when studying this histone modification?

When faced with conflicting data regarding H2B K23 mono-methylation patterns or functions, researchers should consider:

• Antibody Validation: Confirm antibody specificity through peptide competition assays and knockout/knockdown controls

• Cellular Context: Different cell types may exhibit different modification patterns and functional outcomes

• Temporal Dynamics: Consider the dynamic nature of histone modifications and potential cell cycle dependencies

• Technical Approaches: Use orthogonal techniques (mass spectrometry, genomics, microscopy) to validate findings

• Biological Replicates: Increase the number of biological replicates to strengthen statistical power

Many conflicting results in epigenetic research stem from differences in experimental conditions, cell states, or technical approaches. Careful documentation of all experimental variables is essential for resolving such conflicts.

How does this antibody perform in multiplexed detection systems?

For researchers interested in simultaneous detection of multiple histone modifications:

• Sequential Immunofluorescence: Perform sequential staining with careful blocking between steps

• Mass Cytometry: Consider CyTOF approaches for single-cell analysis of multiple modifications

• Multiplex Western Blotting: Use different fluorophores for simultaneous detection of different histone marks

• Barcoded ChIP-seq: Implement barcoding strategies for multiplexed ChIP-seq experiments

When designing multiplexed experiments, careful consideration of antibody species, isotypes, and potential cross-reactivity is essential for accurate data interpretation.

What emerging technologies can enhance studies using this antibody?

Several cutting-edge technologies can significantly enhance research using the Mono-methyl-HIST1H2BC (K23) Antibody:

• CUT&RUN/CUT&Tag: Higher resolution alternatives to ChIP-seq with lower background

• Single-cell Epigenomics: Mapping modification patterns at single-cell resolution

• Live-cell Imaging: Development of modification-specific intrabodies for dynamic studies

• Proximity Ligation Assays: Detecting co-occurrence of multiple modifications at specific loci

• CRISPR Epigenome Editing: Targeted manipulation of H2B K23 methylation states

These emerging approaches offer opportunities to address previously intractable questions about the spatial and temporal dynamics of histone modifications in diverse biological contexts.

How can this modification be studied in the context of disease models?

Researchers investigating the role of H2B K23 mono-methylation in disease contexts should consider:

• Patient-derived Samples: Compare modification patterns in healthy versus diseased tissues

• Disease-relevant Cell Lines: Analyze modification changes in disease models under various conditions

• Pharmacological Modulation: Assess effects of epigenetic drugs on this modification

• Genetic Models: Create genetic models with altered methyltransferase/demethylase activities

• Clinical Correlations: Correlate modification levels with disease progression or treatment response

Understanding how alterations in this histone modification contribute to disease pathogenesis could reveal new diagnostic markers or therapeutic targets for epigenetic intervention.