Mono-methyl-HIST1H2BC (K116) Antibody

What is the biological significance of histone H2B mono-methylation at K116?

Mono-methylation of histone H2B at lysine 116 (K116) represents an important post-translational modification involved in chromatin structure regulation. This specific modification influences DNA accessibility to cellular machinery requiring DNA as a template. Histone H2B serves as a core component of nucleosomes, which wrap and compact DNA into chromatin. The mono-methylation at K116 participates in the complex "histone code" that regulates transcription, DNA repair, replication, and chromosomal stability . Beyond its nuclear functions, histone H2B has demonstrated broad antibacterial activity and may contribute to the formation of functional antimicrobial barriers in colonic epithelium and amniotic fluid .

How does Anti-Histone H2B (mono methyl K116) antibody compare to other histone modification antibodies?

The Anti-Histone H2B (mono methyl K116) antibody is a highly specific tool for detecting a single post-translational modification. Unlike some histone antibodies that show significant cross-reactivity with multiple modifications, properly validated mono methyl K116 antibodies demonstrate high specificity in peptide array testing. For example, when tested against 501 different modified and unmodified histone peptides, the EPR17700 clone shows specific binding to its target epitope . This specificity contrasts with some other histone antibodies that may exhibit cross-reactivity with different modifications, such as antibody #5 directed against H3K4me3 that shows weak binding to peptides containing H3T3ph and cross-reactivity with H4K20me3 .

What experimental applications is the Anti-Histone H2B (mono methyl K116) antibody suitable for?

The Anti-Histone H2B (mono methyl K116) antibody demonstrates versatility across multiple experimental platforms. It has been validated for:

• Western blotting (WB): Successfully detects a 14 kDa band in whole cell lysates from multiple species

• Immunocytochemistry/Immunofluorescence (ICC/IF): Produces clear nuclear staining in fixed and permeabilized cells

• Peptide arrays (PepArr): Shows specific binding in high-throughput peptide screening

• Immunohistochemistry on paraffin-embedded sections (IHC-P): Effective for tissue section analysis

The antibody has demonstrated reactivity with human, mouse, and rat samples, making it valuable for comparative studies across species . When using this antibody for Western blotting, researchers should note that signal intensity may vary between cell types, with some cell lines (like HeLa) showing weaker signals than others (like NIH/3T3) .

How can I validate the specificity of Anti-Histone H2B (mono methyl K116) antibody in my experimental system?

Thorough validation of antibody specificity is crucial for accurate interpretation of histone modification studies. A comprehensive validation approach should include:

• Peptide competition assays: Pre-incubate the antibody with increasing concentrations of the specific mono-methylated K116 peptide before application to your samples. A specific antibody will show diminished signal when pre-bound to its target epitope.

• Peptide array analysis: Test antibody binding against a panel of modified and unmodified histone peptides. As demonstrated with the EPR17700 clone, this approach can reveal both specific binding to the target and any potential cross-reactivity .

• Genetic validation: Use cell lines with mutations in methyltransferases responsible for H2B K116 mono-methylation or employ CRISPR/Cas9 to modify the K116 residue to arginine (which cannot be methylated).

• Mass spectrometry correlation: Compare antibody-based detection methods with mass spectrometry analysis of histone modifications to confirm specificity.

• Positive and negative controls: Include samples with known high and low levels of H2B K116 mono-methylation in each experiment, similar to the stronger signals observed in NIH/3T3 cells compared to HeLa cells .

What are the optimal conditions for detecting mono-methyl-H2B K116 in different experimental contexts?

Optimization strategies vary by experimental application:

For Western blotting:

• Use 5% BSA/TBST as blocking and dilution buffer to minimize background

• Recommended dilution: 1/5000

• Load approximately 10 μg of whole cell lysate per lane

• Use enhanced chemiluminescence (ECL) detection with sensitivity adjusted to sample type (higher sensitivity may be required for samples with lower expression)

For Immunofluorescence:

• Fix cells with 4% paraformaldehyde

• Permeabilize with 0.1% Triton X-100

• Recommended dilution: 1/2000

• Include appropriate nuclear counterstain (e.g., DAPI)

• Include controls for antibody specificity

For Immunohistochemistry:

• Perform heat-mediated antigen retrieval with Tris/EDTA buffer (pH 9.0)

• Recommended dilution: 1/2000

• Counter-stain with hematoxylin for context

• Include negative controls (primary antibody substituted with PBS)

How do modifications in adjacent residues affect the detection of mono-methyl-H2B K116?

The presence of modifications on residues adjacent to or near K116 can significantly impact antibody recognition. This epitope occlusion phenomenon has been documented with various histone antibodies. For example, antibody #5 directed against H3K4me3 shows weak binding when H3T3ph is present (false negatives) .

When investigating mono-methyl-H2B K116, consider these approaches:

• Utilize peptide arrays containing combination modifications to test for potential epitope occlusion

• Employ sequential immunoprecipitation with antibodies against different modifications

• Consider mass spectrometry analysis to identify co-occurring modifications

• Compare results from different antibody clones that may have different sensitivities to adjacent modifications

Adjacent modifications can lead to both false negatives (when the modification prevents antibody binding) and false positives (when the antibody cross-reacts with similar modifications) . This underscores the importance of comprehensive validation and control experiments.

What are the best practices for sample preparation when detecting mono-methyl-H2B K116?

Optimal sample preparation is crucial for accurate detection of histone modifications:

• Histone extraction protocols:

  • Use specialized histone extraction buffers containing histone deacetylase and demethylase inhibitors

  • For acid extraction, use 0.2N HCl or 0.4N H₂SO₄ to efficiently extract histones

  • For whole cell lysates, include inhibitors of proteases and phosphatases

• Preservation of modifications:

  • Add modification-preserving agents: sodium butyrate (10 mM) for acetylation, sodium fluoride (5-10 mM) for phosphorylation

  • Process samples quickly and maintain cold temperatures throughout

  • For tissue samples, snap-freeze immediately after collection

• Buffer considerations:

  • For Western blotting, prepare samples in buffers containing SDS and reducing agents

  • For immunofluorescence, fix with formaldehyde to preserve nuclear structure

  • For IHC-P, perform heat-mediated antigen retrieval with Tris/EDTA buffer (pH 9.0)

• Control samples:

  • Include known positive controls (e.g., NIH/3T3 cells) that show strong mono-methyl-H2B K116 signal

  • Consider cell types with varying expression levels for calibration (HeLa cells show weaker signals than NIH/3T3)

How can I troubleshoot weak or non-specific signals when working with mono-methyl-H2B K116 antibody?

When encountering signal issues, consider these troubleshooting approaches:

For weak signals:

• Decrease antibody dilution while monitoring background

• Extend primary antibody incubation time (overnight at 4°C)

• Use more sensitive detection systems (5-fold more sensitive ECL substrate may be required for some samples)

• Increase protein loading (while ensuring even loading with control antibodies)

• Verify preservation of modifications during sample preparation

For non-specific signals:

• Increase antibody dilution to reduce background (test serial dilutions)

• Optimize blocking conditions (5% BSA/TBST is recommended)

• Include additional washing steps with increased stringency

• Pre-adsorb antibody with non-specific proteins

• Confirm specificity with peptide competition assays

For inconsistent results:

• Standardize cell culture conditions (passage number, confluence)

• Control for cell cycle effects (synchronize cells if necessary)

• Verify antibody storage conditions and avoid repeated freeze-thaw cycles

• Include internal controls in each experiment

• Consider batch effects from antibody lots

What quantitative methods are most appropriate for analyzing mono-methyl-H2B K116 levels?

Several quantitative approaches can be employed for analyzing mono-methyl-H2B K116 levels:

Western blot densitometry:

• Normalize signal to total H2B levels

• Use linear range of detection for quantification

• Include standard curves with known quantities when possible

• Employ software that corrects for background and saturation

Quantitative immunofluorescence:

• Measure nuclear fluorescence intensity using appropriate imaging software

• Normalize to DAPI or total H2B signal

• Establish consistent threshold settings across experiments

• Analyze sufficient cell numbers for statistical validity (n > 100)

ChIP-seq or CUT&RUN:

• Quantify genome-wide distribution of mono-methyl-H2B K116

• Normalize to input and total H2B ChIP

• Use appropriate peak calling algorithms

• Validate findings at selected loci with ChIP-qPCR

Mass spectrometry:

• Provides absolute quantification of modification levels

• Can identify co-occurring modifications

• Requires specialized equipment and expertise

• Offers unbiased detection independent of antibody specificity

How do I interpret variations in mono-methyl-H2B K116 levels between different cell types?

Variations in mono-methyl-H2B K116 levels between cell types, as observed between NIH/3T3 and HeLa cells , may reflect important biological differences:

• Cell type-specific epigenetic programming:

  • Different cell types maintain distinct epigenetic landscapes

  • Variations may correspond to cell-specific transcriptional programs

  • Consider developmental origins and differentiation state of cell types

• Methodological considerations:

  • Verify that differences are not due to technical variables (extraction efficiency, etc.)

  • Confirm findings with multiple detection methods

  • Normalize to appropriate controls (total H2B, other stable references)

• Biological interpretation approaches:

  • Correlate modification levels with gene expression data

  • Map genome-wide distribution using ChIP-seq

  • Analyze co-occurrence with other histone marks

  • Investigate enzymatic machinery (writers, erasers, readers) expression

• Experimental validation:

  • Manipulate levels using genetic or pharmacological approaches

  • Assess functional consequences of alteration

  • Investigate during differentiation or in response to stimuli

What is the relationship between mono-methyl-H2B K116 and other histone modifications?

Understanding the interplay between mono-methyl-H2B K116 and other modifications requires comprehensive analysis:

• Co-occurrence patterns:

  • Multiple modifications may exist simultaneously on the same or adjacent nucleosomes

  • Some modifications may be mutually exclusive while others co-occur

  • Sequential ChIP or mass spectrometry can identify co-occurrences

• Functional relationships:

  • Primary modifications may facilitate or inhibit subsequent modifications

  • Some modifications function as docking sites for effector proteins

  • Others may directly influence chromatin structure

• Cross-talk with DNA methylation:

  • Investigate correlation between H2B K116me1 and DNA methylation patterns

  • Examine relationship with CpG islands and gene regulatory elements

• Data analysis approaches:

  • Generate heatmaps of modification co-occurrence

  • Conduct principal component analysis of multiple modifications

  • Develop predictive models of modification patterns

  • Compare with publicly available datasets for other cell types

How can I integrate mono-methyl-H2B K116 data with other genomic and epigenomic datasets?

Integration of mono-methyl-H2B K116 data with other genomic information provides comprehensive insights:

Data integration strategies:

Advanced computational approaches:

• Machine learning algorithms to predict functional elements based on modification patterns

• Network analysis to identify regulatory hubs

• Integrative genomics viewer (IGV) for visualization of multiple data types

• Regulatory circuit reconstruction using modification data as inputs

These integrative approaches help place mono-methyl-H2B K116 within the broader context of epigenetic regulation and gene expression control mechanisms.