HIST1H2BC (Ab-11) Antibody

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

HIST1H2BC (Histone H2B type 1-C/E/F/G/I) is a member of the histone H2B family that plays a fundamental role in packaging DNA into chromatin structure. This histone variant contributes significantly to nucleosome formation, where DNA wraps around histone octamers to form the basic unit of chromatin. HIST1H2BC functions extend beyond structural roles to include active participation in gene expression regulation and DNA repair processes. The protein achieves this by influencing chromatin accessibility to transcription factors and DNA repair machinery through post-translational modifications and structural arrangements. These functions make HIST1H2BC essential for proper gene regulation and genomic stability maintenance .

What are the key specifications of the HIST1H2BC (Ab-11) Antibody?

The HIST1H2BC (Ab-11) Antibody (PACO60476) is a rabbit-derived polyclonal antibody that specifically targets the region surrounding lysine 11 (Lys-11) in human Histone H2B type 1-C/E/F/G/I. This antibody is supplied as a liquid formulation (50μl) in a storage buffer containing 0.03% Proclin 300, 50% Glycerol, and 0.01M PBS at pH 7.4. Its specificity is derived from the immunogen consisting of a peptide sequence around the Lys-11 site. The antibody belongs to the IgG isotype, is non-conjugated, and has undergone antigen affinity purification to ensure high specificity. Its demonstrated reactivity is primarily with human samples, making it particularly valuable for studying human histone biology .

What experimental applications is this antibody validated for?

The HIST1H2BC (Ab-11) Antibody has been validated for multiple experimental applications, with specific recommended dilution parameters for each technique:

While not explicitly stated in the search results, researchers commonly adapt such antibodies for Western blotting applications as well, typically starting with manufacturer-recommended dilutions. When using this antibody for immunohistochemistry, researchers should note that validation has been performed on a Leica BondTM system with specific antigen retrieval conditions (high-pressure citrate buffer, pH 6.0) .

How does histone modification affect HIST1H2BC function in chromatin dynamics?

Histone modifications, particularly ubiquitination, significantly impact HIST1H2BC function and chromatin dynamics. Research indicates that histone H2A lysine 119 ubiquitination (H2AK119ub) levels are regulated by deubiquitinating enzymes like BAP1 (BRCA1-associated protein 1), which directly affects B-cell activation and antibody production. When BAP1 is lost in activated B cells, there is a genome-wide dysregulation of H2AK119ub levels, resulting in altered gene expression patterns .

While this mechanism has been established for H2A, similar regulatory processes likely apply to HIST1H2BC, where post-translational modifications alter nucleosome dynamics and accessibility. Such modifications create a "histone code" that influences transcription factor binding, chromatin remodeling, and ultimately gene expression. When studying HIST1H2BC with the Ab-11 antibody, researchers should consider how these modifications might affect epitope accessibility and antibody binding, particularly in experiments examining differential gene expression or chromatin immunoprecipitation .

What controls and validation steps should be implemented when using this antibody in research?

Implementing rigorous controls and validation steps is crucial for generating reliable data with the HIST1H2BC (Ab-11) Antibody:

• Positive Control: Include samples known to express HIST1H2BC (human small intestine tissue sections have been validated) .

• Negative Controls:

  • Omit primary antibody but include all other reagents to detect non-specific binding of secondary antibodies

  • Use samples from knockout models (if available) or tissues known not to express the target

  • Include isotype control antibodies (rabbit IgG) to detect non-specific binding

• Antibody Validation:

  • Peptide competition assay: Pre-incubate antibody with immunizing peptide to demonstrate binding specificity

  • Western blot validation: Confirm antibody detects a protein of the expected molecular weight (approximately 14 kDa for HIST1H2BC)

  • Cross-validation with different antibody clones targeting other epitopes of HIST1H2BC

  • Correlation of protein detection with mRNA expression data

• Protocol Optimization:

  • Titrate antibody concentrations to determine optimal signal-to-noise ratio

  • Test different antigen retrieval methods for IHC/IF applications

  • Validate signal specificity across multiple experimental replicates and conditions

How might aberrant HIST1H2BC expression contribute to disease pathogenesis?

Aberrant expression of HIST1H2BC has been linked to various disease states, particularly cancer, though the mechanisms are complex and multifaceted. As a core histone protein involved in chromatin packaging, altered HIST1H2BC levels or modifications can disrupt normal gene expression patterns, potentially contributing to pathogenesis through several mechanisms:

• Dysregulated Gene Expression: Abnormal HIST1H2BC expression may alter chromatin accessibility, leading to inappropriate activation or silencing of genes involved in cell cycle regulation, DNA repair, or apoptosis.

• Genomic Instability: Disruptions in histone-DNA interactions can compromise genomic integrity, potentially increasing mutation rates and chromosomal abnormalities.

• Epigenetic Reprogramming: Changes in histone modification patterns, including those involving HIST1H2BC, can establish aberrant epigenetic states that promote disease progression.

• Altered DNA Damage Response: HIST1H2BC plays a role in DNA repair processes; its dysregulation may impair the cell's ability to respond to DNA damage, contributing to genomic instability.

Researchers using the HIST1H2BC (Ab-11) Antibody can investigate these disease connections by comparing HIST1H2BC expression and modification patterns between normal and diseased tissues, correlating expression with disease progression markers, or studying the effects of experimental manipulation of HIST1H2BC expression on cellular phenotypes .

What protocol optimizations are recommended for immunohistochemistry applications?

For optimal immunohistochemistry (IHC) results with the HIST1H2BC (Ab-11) Antibody, researchers should consider the following protocol optimizations:

• Tissue Preparation and Fixation:

  • Use freshly fixed tissues when possible (10% neutral buffered formalin)

  • Limit fixation time to 24-48 hours to prevent over-fixation and epitope masking

  • Process tissues promptly to paraffin embedding

• Antigen Retrieval:

  • Implement high-pressure antigen retrieval in citrate buffer (pH 6.0) as validated with this antibody

  • Optimize retrieval time (typically 10-20 minutes) based on tissue type and fixation conditions

• Blocking and Antibody Incubation:

  • Block with 10% normal goat serum for 30 minutes at room temperature

  • Dilute primary antibody in 1% BSA solution at 1:10-1:100 (start with 1:20 for initial testing)

  • Incubate primary antibody at 4°C overnight for optimal binding

  • Use a biotinylated secondary antibody system followed by HRP conjugation for visualization

• Signal Development and Counterstaining:

  • Use an HRP-conjugated SP system for detection

  • Optimize DAB development time to achieve optimal signal-to-noise ratio

  • Consider light hematoxylin counterstaining to visualize nuclear morphology without obscuring the specific signal

• System Compatibility:

  • The antibody has been validated on a Leica BondTM automated system, which may provide more consistent results than manual staining

  • When using other automated platforms, validate and optimize conditions accordingly

What are effective approaches for troubleshooting weak or nonspecific signals?

Researchers commonly encounter weak signals or nonspecific background when using antibodies like HIST1H2BC (Ab-11). Here are effective troubleshooting strategies:

For Weak Signals:

• Antibody Concentration: Increase antibody concentration incrementally while staying within recommended ranges (1:10-1:100 for IHC).

• Antigen Retrieval Enhancement: Extend retrieval time or test alternative methods (EDTA-based buffers may work better for some epitopes).

• Incubation Time: Extend primary antibody incubation (up to 48 hours at 4°C) or secondary antibody incubation (60-90 minutes).

• Detection System: Switch to a more sensitive detection system, such as tyramide signal amplification.

• Sample Preservation: Ensure samples were properly fixed and stored to preserve epitope integrity.

For Nonspecific Background:

• Additional Blocking: Implement avidin/biotin blocking steps if using biotinylated detection systems.

• Buffer Optimization: Add 0.1-0.3% Triton X-100 to reduce nonspecific hydrophobic interactions.

• Antibody Dilution: Increase antibody dilution systematically while extending incubation time.

• Washing Steps: Increase the number and duration of washing steps (using TBS-T or PBS-T).

• Secondary Antibody Cross-Reactivity: Test alternative secondary antibodies if cross-reactivity is suspected.

For Inconsistent Results:

• Batch Processing: Process all experimental samples simultaneously to minimize technical variation.

• Standard Curves: Include calibration samples with known expression levels.

• Automated Systems: Consider using automated staining platforms like the Leica BondTM system for greater consistency .

How can researchers effectively design experiments to study HIST1H2BC in relation to chromatin modifications?

Designing robust experiments to study HIST1H2BC in relation to chromatin modifications requires strategic approaches that combine multiple techniques:

• ChIP-Seq Analysis:

  • Use HIST1H2BC (Ab-11) Antibody for chromatin immunoprecipitation followed by sequencing

  • Compare HIST1H2BC binding patterns with maps of known histone modifications (H3K27ac, H3K4me3, etc.)

  • Include negative controls (IgG) and positive controls (pan-H2B antibodies)

• Sequential ChIP (Re-ChIP):

  • Perform first immunoprecipitation with the HIST1H2BC (Ab-11) Antibody

  • Follow with a second immunoprecipitation using antibodies against specific histone modifications

  • This approach identifies genomic regions where HIST1H2BC coincides with particular modifications

• Correlative Microscopy:

  • Combine immunofluorescence using HIST1H2BC (Ab-11) Antibody with antibodies against histone-modifying enzymes

  • Quantify co-localization coefficients to establish spatial relationships

  • Consider super-resolution microscopy for detailed chromatin structure analysis

• Functional Studies with Genetic Manipulation:

  • Design experiments with BAP1 knockout/knockdown systems to study effects on HIST1H2BC and H2AK119ub levels

  • Compare chromatin accessibility (ATAC-seq) with HIST1H2BC distribution and modification patterns

  • Correlate changes in HIST1H2BC occupancy with alterations in gene expression

• Extraction Fractionation Experiments:

  • Separate chromatin into fractions based on compaction state

  • Analyze HIST1H2BC levels and modifications across different chromatin fractions

  • Correlate with gene expression in associated regions

These experimental designs should incorporate appropriate controls and consider the technical limitations of the HIST1H2BC (Ab-11) Antibody, including its specificity for human samples and optimal working dilutions for each technique .

How does HIST1H2BC interact with the cellular machinery during B-cell activation and antibody production?

Recent research has elucidated critical relationships between histone biology and B-cell function that are relevant to understanding HIST1H2BC's role. While specific HIST1H2BC interactions are still being characterized, studies on related histone modifications provide valuable insights:

Histone deubiquitination by BAP1 plays a crucial role in regulating B-cell activation and antibody production. BAP1 regulates histone H2AK119ub levels, which directly impacts genome-wide gene expression patterns essential for B-cell proliferation and function. When BAP1 is deleted in activated B cells, there is severe impairment of antibody production, along with altered dynamics of germinal center B cells, memory B cells, and plasma cells .

Given the structural and functional similarities between histone variants, HIST1H2BC likely undergoes similar regulatory processes during B-cell activation. The HIST1H2BC protein may participate in:

• Transcriptional Regulation: Modulating accessibility of genes required for B-cell activation and differentiation

• Class Switch Recombination: Contributing to chromatin reorganization necessary for antibody class switching

• Cell Proliferation Control: Regulating cell cycle genes essential for B-cell clonal expansion

• Epigenetic Memory: Establishing heritable chromatin states in memory B cells

Researchers can use the HIST1H2BC (Ab-11) Antibody to investigate these interactions by:

• Tracking HIST1H2BC levels and modifications during different stages of B-cell activation

• Comparing HIST1H2BC distribution in naive versus activated B cells

• Examining co-localization with B-cell-specific transcription factors

• Correlating HIST1H2BC modifications with antibody production efficiency

What are the latest methodological advances in studying histone variants like HIST1H2BC?

Recent methodological advances have significantly enhanced researchers' ability to study histone variants like HIST1H2BC with unprecedented precision:

• Mass Spectrometry-Based Approaches:

  • Middle-down and top-down proteomics now allow identification of combinatorial histone modifications

  • Cross-linking mass spectrometry (XL-MS) can map protein-protein interactions within chromatin complexes

  • Quantitative proteomics enables precise measurement of histone variant stoichiometry in different cellular states

• Advanced Imaging Techniques:

  • Live-cell super-resolution microscopy permits real-time tracking of histone dynamics

  • Correlative light and electron microscopy (CLEM) connects histone distribution with ultrastructural context

  • Single-molecule tracking reveals kinetics of histone exchange and residence time

• Genomic and Epigenomic Integration:

  • CUT&RUN and CUT&Tag provide higher signal-to-noise ratio than traditional ChIP

  • Combinatorial indexed ChIP-seq allows multiplexed analysis of histone variants

  • Single-cell epigenomic profiling reveals cell-to-cell variation in histone variant distribution

• CRISPR-Based Technologies:

  • Prime editing enables precise modification of histone genes with minimal off-target effects

  • CRISPRi/CRISPRa systems allow temporal control of histone variant expression

  • CRISPR screens identify factors affecting histone variant incorporation and function

• Computational Methods:

  • Machine learning algorithms detect subtle patterns in histone variant distribution

  • Integrative multi-omics approaches correlate histone variants with gene expression and chromatin structure

  • Molecular dynamics simulations predict functional effects of histone variant incorporation

Researchers can leverage these advances alongside antibodies like HIST1H2BC (Ab-11) to gain deeper insights into chromatin biology and gene regulation .

How might understanding HIST1H2BC function contribute to therapeutic approaches for diseases like cancer?

Understanding HIST1H2BC function has significant implications for developing novel therapeutic approaches, particularly for cancer:

• Biomarker Development:

  • Aberrant HIST1H2BC expression has been linked to cancer development

  • The HIST1H2BC (Ab-11) Antibody could be employed to develop diagnostic or prognostic tests based on HIST1H2BC expression patterns or modifications

  • Characterizing HIST1H2BC alterations in different cancer types may help stratify patients for personalized treatment approaches

• Epigenetic Drug Targeting:

  • Enzymes that modify HIST1H2BC (writers, readers, and erasers) represent potential therapeutic targets

  • Understanding how HIST1H2BC contributes to gene regulation may reveal vulnerabilities in cancer cells that depend on specific epigenetic states

  • Combination therapies targeting both HIST1H2BC modifications and related pathways could enhance treatment efficacy

• Immunotherapy Applications:

  • Given histone modifications' role in B-cell function, understanding HIST1H2BC could improve our ability to modulate immune responses

  • Cancer immunotherapies might be enhanced by targeting pathways that regulate HIST1H2BC in immune cells

  • HIST1H2BC-derived neoantigens could potentially serve as targets for cancer vaccines

• Precision Medicine Approaches:

  • Characterizing patient-specific alterations in HIST1H2BC and related histone proteins may enable more targeted interventions

  • Functional studies using patient-derived cells and the HIST1H2BC (Ab-11) Antibody could help predict treatment responses

  • Computational models incorporating HIST1H2BC data may improve patient stratification for clinical trials

These therapeutic applications highlight the importance of rigorous basic research on histone biology using tools like the HIST1H2BC (Ab-11) Antibody, which provides a means to investigate these complex relationships in human samples .

What emerging research questions about HIST1H2BC remain to be addressed?

Despite advances in our understanding of HIST1H2BC, several critical research questions remain that researchers using the HIST1H2BC (Ab-11) Antibody could address:

• Variant-Specific Functions: How do the functions of HIST1H2BC differ from other H2B variants, and what mechanisms confer this specificity?

• Tissue-Specific Regulation: Does HIST1H2BC expression and modification vary systematically across different human tissues and cell types, and what functional consequences result from these variations?

• Disease Progression Dynamics: How do patterns of HIST1H2BC modification change during disease progression, particularly in cancer and immune disorders?

• Regulatory Networks: What upstream signaling pathways and transcription factors control HIST1H2BC expression and incorporation into chromatin?

• Therapeutic Modulation: Can HIST1H2BC be specifically targeted for therapeutic intervention, and what approaches might prove most effective?

• Technological Development: How can detection methods for HIST1H2BC and its modifications be improved for greater sensitivity and specificity?

Addressing these questions will require innovative experimental approaches, careful controls, and judicious use of research tools like the HIST1H2BC (Ab-11) Antibody .