HIST1H2BC (Ab-85) Antibody

What is HIST1H2BC and what role does it play in cellular functions?

HIST1H2BC is a core component of nucleosomes, the fundamental unit of chromatin structure in eukaryotic cells. As part of the histone H2B family, HIST1H2BC plays critical roles in packaging DNA into chromatin, thereby controlling DNA accessibility to various cellular machineries . This histone variant participates in several essential cellular processes:

• Transcription regulation through modulation of DNA accessibility to transcription factors

• DNA repair via chromatin remodeling at damage sites

• DNA replication through controlled unwinding of the double helix

• Maintenance of chromosomal stability and structural integrity

The functional significance of HIST1H2BC extends beyond structural roles, as it undergoes various post-translational modifications that constitute part of the "histone code." These modifications, including acetylation, methylation, phosphorylation, and ubiquitination, regulate DNA accessibility through complex biochemical signaling mechanisms . The HIST1H2BC (Ab-85) Antibody specifically targets the region around lysine 85, allowing researchers to study this particular histone variant and its role in chromatin organization and gene expression regulation.

What applications has the HIST1H2BC (Ab-85) Antibody been validated for?

The HIST1H2BC (Ab-85) Antibody has been validated for multiple research applications, with specific recommended dilution ranges for optimal results:

For effective results in these applications:

• For ELISA applications: Begin with a middle-range dilution (1:5000) and adjust based on signal intensity. Ensure proper blocking (typically 5% BSA or milk) to minimize background signal .

• For IHC applications: Start with a 1:50 dilution, employing appropriate antigen retrieval methods to expose the epitope. Heat-induced epitope retrieval in citrate buffer (pH 6.0) is typically effective for histone proteins .

While not specifically validated in the provided data, related HIST1H2BC antibodies have been successfully used for Western blotting and immunoprecipitation applications. If attempting Western blotting, a starting dilution of 1:100-1:1000 would be recommended, with expected band size around 14-15 kDa .

What are the optimal storage conditions for maintaining antibody activity?

For maintaining optimal HIST1H2BC (Ab-85) Antibody activity over time:

• Long-term storage: The recommended storage temperature is -20°C, which preserves antibody functionality and prevents degradation .

• Storage buffer composition: The antibody is supplied in a protective buffer containing 50% glycerol, 0.03% Proclin 300, and 0.01M PBS at pH 7.4, which helps maintain stability during freeze-thaw cycles .

• Aliquoting recommendations: Upon first thawing, divide the antibody into small aliquots to minimize repeated freeze-thaw cycles, which can cause protein denaturation and reduced activity.

Regarding working dilutions:

• Fresh preparation: For immunohistochemistry applications, it is generally recommended to prepare fresh working dilutions when applying the primary antibody rather than storing diluted solutions .

• Short-term storage: If storage of working dilutions is necessary, keep at 4°C for no more than 1-2 weeks, with awareness that this may affect antibody performance .

• Avoid freezing dilutions: Once working dilutions for IHC have been generated, it is not recommended to freeze these dilutions or store them in the refrigerator for prolonged periods .

To monitor antibody performance over time, include positive control samples in experiments to detect any potential loss of activity during storage.

What is the species reactivity of the HIST1H2BC (Ab-85) Antibody?

The HIST1H2BC (Ab-85) Antibody has been specifically developed and validated for reactivity with human samples. The antibody was raised against a peptide sequence surrounding lysine 85 derived from Human Histone H2B type 1-C/E/F/G/I .

Key specificity considerations include:

• The antibody recognizes human HIST1H2BC protein (UniProt accession: P62807) .

• Due to the highly conserved nature of histones across mammalian species, there may be potential cross-reactivity with mouse, rat, or other mammalian HIST1H2BC variants, although this would require empirical verification.

• The antibody may recognize several histone H2B variants due to sequence homology, as indicated by its recognition of Histone H2B type 1-C/E/F/G/I variants. This is reflected in its synonym designations: H2BFL, H2BFH, H2BFG, H2BFA, and H2BFK .

When using this antibody, researchers should be aware that the immunogen sequence was specifically derived from human samples, and the validated reactivity is against human proteins . To ensure specificity in experiments, inclusion of appropriate negative controls (such as isotype controls) and positive controls (human cell lines known to express HIST1H2BC) is recommended.

How do I optimize immunohistochemistry protocols for HIST1H2BC (Ab-85) Antibody?

Optimizing IHC protocols for HIST1H2BC (Ab-85) Antibody requires careful attention to several key parameters:

Sample Preparation and Antigen Retrieval:

• Fixation: Standard 10% neutral buffered formalin fixation for 24-48 hours provides consistent results for histone proteins.

• Sectioning: 4-6 μm sections on positively charged slides prevent tissue detachment during high-temperature antigen retrieval.

• Antigen retrieval: For histone proteins like HIST1H2BC, heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) is typically effective. Consider extending retrieval time to 20-30 minutes to ensure adequate exposure of nuclear epitopes .

Blocking and Antibody Incubation:

• Blocking: Use 5-10% normal serum (from the secondary antibody species) with 1% BSA in PBS for 1-2 hours at room temperature to minimize background staining.

• Primary antibody: Begin with 1:50 dilution (as recommended) in blocking buffer and incubate overnight at 4°C for optimal binding .

• Secondary antibody: Use species-appropriate HRP-conjugated secondary antibody at manufacturer's recommended dilution (typically 1:200-1:500).

Optimization Strategy:

• Perform a dilution series (e.g., 1:10, 1:50, 1:100) to determine optimal concentration for your specific tissue type .

• Compare different antigen retrieval methods if standard citrate buffer yields suboptimal results.

• Test different incubation times and temperatures for primary antibody.

• Include positive control tissues known to express HIST1H2BC (most proliferating tissues and cell lines like HeLa can serve as positive controls) .

Troubleshooting Common Issues:

• High background: Increase blocking time, reduce antibody concentration, or add 0.1-0.3% Triton X-100 to permeabilize nuclear membranes more effectively.

• Weak signal: Enhance antigen retrieval time, increase antibody concentration, or extend incubation time.

• Non-specific binding: Use more stringent washing (add 0.05% Tween-20 to wash buffers) and ensure adequate blocking.

What are the differences between acetylated and non-acetylated HIST1H2BC antibodies?

Understanding the differences between antibodies targeting acetylated HIST1H2BC (such as Acetyl-HIST1H2BC (K85)) and non-acetylated HIST1H2BC is crucial for epigenetic research:

Functional implications of acetylation:
Acetylation of H2B at K85 typically correlates with transcriptionally active regions of the genome . When lysine residues are acetylated, the positive charge is neutralized, which weakens histone-DNA interactions and leads to a more open chromatin structure, facilitating transcription factor binding and gene expression.

Methodological considerations:

• For comprehensive epigenetic studies, using both antibodies in parallel can provide insights into the relationship between histone modification and gene expression patterns.

• When performing ChIP experiments, acetylation-specific antibodies like Acetyl-HIST1H2BC (K85) are particularly valuable for identifying regions of active transcription .

• For dual immunofluorescence studies, ensure that the antibodies are raised in different host species to avoid cross-reactivity of secondary antibodies.

Research applications:

• The non-acetylated antibody is useful for baseline studies of nucleosome density and general chromatin organization .

• The acetylation-specific antibody enables the study of dynamic gene regulation, cell differentiation, and responses to environmental stimuli through epigenetic mechanisms .

How can I use HIST1H2BC antibodies in conjunction with other epigenetic markers?

Combining HIST1H2BC antibodies with other epigenetic markers provides a comprehensive view of chromatin states and regulatory mechanisms. This multi-marker approach enables researchers to understand the complex interplay between different histone modifications and their functional outcomes.

Co-immunostaining Strategies:

• For immunofluorescence: Use HIST1H2BC (Ab-85) Antibody in combination with antibodies against other histone marks such as H3K4me3 (active promoters), H3K27me3 (repressed regions), or H3K9ac (active enhancers).

• Ensure primary antibodies are raised in different host species (e.g., rabbit anti-HIST1H2BC with mouse anti-H3K4me3) to allow for specific secondary antibody detection.

• Implement sequential staining protocols if using antibodies from the same species, with careful blocking between rounds.

ChIP-seq Integration:

• Perform parallel ChIP-seq experiments with HIST1H2BC antibodies and other histone modification antibodies to create comprehensive epigenetic maps .

• Integrate datasets to identify genomic regions where HIST1H2BC co-localizes with specific modifications.

• Correlate these patterns with gene expression data (RNA-seq) to establish functional relationships.

Epigenetic Profiling in Disease Models:

• Examine changes in HIST1H2BC distribution alongside other histone marks in disease states, particularly in cancer where epigenetic dysregulation is common.

• Investigate the role of BAP1 (a deubiquitinating enzyme) in regulating genome-wide histone modification landscapes, including effects on H2AK119ub and their relationship to HIST1H2BC distribution .

• Study how these epigenetic patterns correlate with gene expression changes in disease progression.

Practical Implementation:

• For flow cytometry applications, design multi-parameter panels that include HIST1H2BC alongside other nuclear markers.

• For mass spectrometry approaches, consider using antibody-based enrichment of HIST1H2BC-containing nucleosomes followed by analysis of associated modifications.

• For spatial analysis, implement advanced imaging techniques like super-resolution microscopy to visualize the nuclear distribution of different histone marks in relation to HIST1H2BC.

By combining HIST1H2BC antibodies with other epigenetic markers, researchers can develop a more comprehensive understanding of chromatin regulation and its impact on cellular functions in both normal and pathological states.

How can I implement ChIP protocols using HIST1H2BC antibodies for epigenetic profiling?

Implementing Chromatin Immunoprecipitation (ChIP) protocols with HIST1H2BC antibodies requires careful optimization to achieve sensitive and specific mapping of histone occupancy and modifications:

Sample Preparation and Chromatin Extraction:

• Crosslinking Optimization:

  • For histone studies, use 1% formaldehyde for 10 minutes at room temperature.

  • For detecting more transient interactions, consider dual crosslinking with EGS (ethylene glycol bis(succinimidyl succinate)) before formaldehyde.

  • Quench with glycine (final concentration 0.125M) for 5 minutes.

• Chromatin Shearing:

  • Target fragment size: 200-500 bp for high-resolution mapping.

  • Sonication: Typically 10-15 cycles (30s ON/30s OFF) with Bioruptor or similar device.

  • Enzymatic digestion: Consider Micrococcal Nuclease (MNase) digestion for nucleosome-resolution studies.

  • Verify fragment size by agarose gel electrophoresis before proceeding.

Immunoprecipitation with HIST1H2BC Antibodies:

• Antibody Selection:

  • For total HIST1H2BC: Use non-modified HIST1H2BC (Ab-85) Antibody.

  • For specific modifications: Use modification-specific antibodies like Acetyl-HIST1H2BC (K85) .

  • Perform preliminary testing including IP-Western blot to validate antibody efficiency.

• ChIP Protocol Optimization:

  • Pre-clearing: 1-2 hours with protein A/G beads to reduce background.

  • Antibody incubation: Overnight at 4°C with 2-5 μg antibody per ChIP reaction.

  • Wash buffers: Include low salt, high salt, LiCl, and TE washes to reduce non-specific binding.

  • Elution: 1% SDS, 0.1M NaHCO₃ at 65°C for efficient release of chromatin.

• Controls:

  • Input control: Reserve 5-10% of chromatin before immunoprecipitation.

  • Negative control: IgG from same species as primary antibody.

  • Positive control: Use established antibodies against H3K4me3 (active promoters) or H3K27me3 (repressed regions).

ChIP-seq Library Preparation and Analysis:

• Library Preparation Considerations:

  • Input requirement: Typically 1-10 ng of ChIP DNA.

  • PCR cycles: Minimize to reduce amplification bias (typically 10-15 cycles).

  • Size selection: 250-600 bp including adapters.

• Data Analysis Pipeline:

  • Alignment: BWA or Bowtie2 to reference genome.

  • Peak calling: MACS2 for broad histone marks with appropriate parameters.

  • Visualization: IGV, UCSC Genome Browser.

  • Integration: Correlate with RNA-seq or other epigenomic data.

Troubleshooting ChIP Experiments:

The Acetyl-HIST1H2BC (K85) antibody has been specifically validated for ChIP applications, making it particularly valuable for mapping acetylation patterns across the genome .

What is the relationship between HIST1H2BC modifications and gene expression regulation?

The relationship between HIST1H2BC modifications and gene expression regulation represents a complex interplay of epigenetic mechanisms that influence chromatin accessibility and transcriptional activity:

Histone Code Interpretation:

• Different modifications on HIST1H2BC (acetylation, methylation, phosphorylation, ubiquitination) serve as recognition sites for specific regulatory proteins .

• H2B acetylation (including at K85) generally correlates with transcriptional activation by reducing the positive charge of histones, weakening DNA-histone interactions, and creating a more open chromatin structure .

• H2B ubiquitination plays a role in transcriptional elongation and can influence methylation of other histones.

Cross-talk with Other Histone Modifications:

• H2B modifications influence the recruitment of chromatin remodeling complexes that can further modify chromatin structure.

• The acetylation state of H2B at specific residues like K85 can affect the binding of bromodomain-containing proteins that recruit transcriptional co-activators.

• Modified HIST1H2BC serves as a platform for assembling regulatory complexes that influence transcription initiation and elongation.

Influence on Nucleosome Dynamics:

• Acetylation of H2B affects nucleosome stability and mobility, influencing the accessibility of DNA to transcription factors.

• Modified H2B can alter the interactions between neighboring nucleosomes, affecting higher-order chromatin structure.

Experimental Approaches to Study These Relationships:

• Integrated ChIP-seq Analysis:

  • Correlate HIST1H2BC modification patterns (using modification-specific antibodies) with transcriptome data (RNA-seq) to identify relationships between specific modifications and gene expression levels.

  • Create genome-wide maps of HIST1H2BC occupancy and modifications in different cellular states.

• Targeted Mutagenesis:

  • Introduce point mutations at specific modification sites (e.g., K85R to prevent acetylation) to assess the functional consequences on gene expression.

  • Use CRISPR/Cas9-mediated genome editing to create cellular models with non-modifiable H2B variants.

• Enzyme Inhibitor Studies:

  • Employ specific inhibitors of histone-modifying enzymes (HDACs, HATs) to manipulate HIST1H2BC modification status and observe effects on gene expression.

Understanding the relationship between HIST1H2BC modifications and gene expression is critical for elucidating the mechanisms of epigenetic regulation in normal development and disease states .

What is the role of HIST1H2BC in B-cell immunity and its connection to BAP1 regulation?

Recent research has revealed important connections between histone regulation, including HIST1H2BC modifications, and B-cell mediated immunity, particularly through the activity of the deubiquitinating enzyme BAP1 (BRCA1-associated protein 1):

BAP1 and Histone Regulation:

B-cell Intrinsic Role of BAP1:

• Research using the Bap1^fl/fl^ Cγ1-cre murine model has demonstrated that B-cell intrinsic loss of BAP1 results in severe defects in antibody production .

• BAP1 deficiency leads to altered dynamics of germinal center B cells, memory B cells, and plasma cells .

• At the cellular level, BAP1 was found to be dispensable for B cell immunoglobulin class switching but resulted in impaired proliferation of activated B cells .

Molecular Mechanisms:

• BAP1 loss leads to genome-wide dysregulation of histone H2AK119ub levels and gene expression .

• These altered histone modification patterns affect the expression of genes critical for B cell activation and function.

• The cross-talk between different histone modifications, including those on HIST1H2BC, creates a complex regulatory network controlling B cell gene expression.

Research Applications:

• HIST1H2BC antibodies can be used in ChIP-seq experiments to map histone occupancy in normal vs. BAP1-deficient B cells.

• Combined analysis of histone modifications and transcriptome data can help identify key regulatory networks affected by BAP1 loss.

• Immunofluorescence studies using HIST1H2BC antibodies can visualize changes in nuclear distribution of histone marks in activated B cells.

Understanding the interplay between HIST1H2BC and other histone modifications in B cells provides insight into the epigenetic regulation of antibody-mediated immunity. Research in this area may lead to new therapeutic approaches for modulating immune responses in various disease contexts .

How can I design multi-parameter flow cytometry experiments incorporating HIST1H2BC antibodies?

Designing multi-parameter flow cytometry experiments that incorporate HIST1H2BC antibodies requires careful consideration of sample preparation, panel design, and analysis strategies:

Sample Preparation for Intracellular Histone Staining:

• Cell Fixation and Permeabilization:

  • Use a two-step protocol for optimal histone detection:

    1. Fix cells with 2-4% paraformaldehyde for 10-15 minutes at room temperature.

    2. Permeabilize with methanol (90% pre-chilled, -20°C for 30 minutes) or specialized nuclear permeabilization buffers.

  • Alternative: Commercial kits designed for intranuclear staining (e.g., Foxp3/Transcription Factor Staining Buffer Sets).

• Blocking and Staining Sequence:

  • Surface marker staining: Perform before fixation using antibodies resistant to fixation/permeabilization.

  • Blocking: 5% serum in permeabilization buffer for 30 minutes.

  • Histone staining: Incubate with HIST1H2BC (Ab-85) Antibody (1:50-1:100) for 45-60 minutes at room temperature.

  • Secondary detection: Use fluorescently labeled anti-rabbit secondary antibody if primary is unconjugated.

Panel Design for Multi-Parameter Analysis:

• B Cell Focused Panel:

  Target	Purpose	Suggested Fluorochrome
  CD19	B cell identification	BV421
  CD27	Memory B cell marker	PE-Cy7
  IgD	Naïve/memory discrimination	FITC
  CD38	Plasma cell/activation marker	APC-Cy7
  HIST1H2BC	Histone detection	PE
  Acetyl-HIST1H2BC (K85)	Acetylation status	APC
  H2AK119ub	BAP1 activity marker	Alexa Fluor 647
  Viability dye	Dead cell exclusion	BV510

• Cell Cycle Analysis Panel:

  Target	Purpose	Suggested Fluorochrome
  HIST1H2BC	Histone detection	PE
  pHistone H3 (Ser10)	Mitosis marker	Alexa Fluor 647
  Ki-67	Proliferation marker	FITC
  DAPI or Hoechst	DNA content	BV421
  Cyclin A	S/G2 phase marker	PE-Cy7

• Controls:

  • Single-stained controls for compensation

  • Fluorescence Minus One (FMO) controls for accurate gating

  • Isotype controls for non-specific binding assessment

  • Positive biological controls (e.g., stimulated cells with known histone modifications)

Optimization Strategies:

• Titration of HIST1H2BC Antibody:

  • Perform serial dilutions (1:25, 1:50, 1:100, 1:200) to determine optimal concentration .

  • Evaluate signal-to-noise ratio at each concentration.

  • Select concentration that maximizes specific signal while minimizing background.

• Cell Cycle Considerations:

  • Histone content and modifications vary throughout the cell cycle.

  • Include DNA content staining (DAPI/Hoechst) to correlate histone status with cell cycle phase.

  • Synchronize cells if studying cell cycle-specific changes in HIST1H2BC modifications.

Advanced Analysis Approaches:

• High-Dimensional Analysis:

  • Implement t-SNE or UMAP dimensionality reduction to visualize complex relationships.

  • Apply clustering algorithms to identify cell populations with distinct histone modification patterns.

  • Consider trajectory analysis to map epigenetic changes during cellular differentiation.

• Single-Cell Correlation Analysis:

  • Correlate HIST1H2BC modifications with surface marker expression to identify cell subsets with unique epigenetic profiles.

  • Link with functional readouts to connect epigenetic state with cell function.

These multi-parameter approaches allow researchers to study the dynamics of HIST1H2BC modifications in specific cell populations and under different biological conditions, particularly in the context of B-cell immunity and BAP1 regulation .