HIST1H2BC (Ab-108) Antibody

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

HIST1H2BC is a histone protein that belongs to the histone cluster 1 H2bc family. It functions as a core component of nucleosomes, playing a crucial role in packaging DNA into chromatin. This protein is specifically involved in gene regulation and chromatin organization processes that influence gene expression and cellular development . HIST1H2BC is part of the larger H2B histone family, which includes 22 genes encoding different H2B variants within the human genome . Fifteen of these variants are found within the HIST1 gene cluster, encoding 11 unique variants, while five are located on the HIST2 cluster and two in the HIST3 cluster . Understanding HIST1H2BC function is particularly relevant for research in epigenetics, cancer biology, and developmental disorders, as it has been implicated in these disease mechanisms .

What are the validated applications for HIST1H2BC (Ab-108) Antibody?

The HIST1H2BC (Ab-108) Antibody (PACO59652) has been validated for multiple research applications with specific recommended dilutions:

This antibody has demonstrated positive Western blot results with several cell lines including Hela, 293, HepG2, HL60, and MCF cell lysates . When conducting these applications, it's essential to optimize the antibody concentration for your specific experimental conditions and cell types.

How should samples be prepared for optimal HIST1H2BC detection?

For effective detection of HIST1H2BC using the Ab-108 antibody, sample preparation protocols should be tailored to the specific application. For Western blotting, cells should be lysed under denaturing conditions that preserve post-translational modifications, particularly if studying ubiquitylation patterns . A recommended approach based on published methods includes:

• Harvest cells at 70-80% confluence (OD 600 of ~0.8-1.2 for yeast studies)

• Perform cell lysis using glass bead disruption in SUTEB buffer (1% SDS, 8M urea, 10mM Tris pH 8.0, 10mM EDTA pH 8.0, 0.01% bromophenol blue)

• Resolve extracts on 15% SDS-polyacrylamide gels

• Transfer proteins to PVDF membranes

• Block with appropriate blocking solution (typically 5% non-fat milk or BSA)

• Probe with HIST1H2BC antibody at recommended dilution (1:100-1:1000)

For immunohistochemistry applications, standard formalin-fixed paraffin-embedded (FFPE) tissue preparation protocols are suitable, with antigen retrieval methods optimized to expose the epitope around the Lys-108 site .

How does HIST1H2BC (Ab-108) Antibody compare with HIST1H2BC (Ab-20) Antibody?

Both antibodies target the same histone protein (HIST1H2BC) but recognize different epitopes, which can provide complementary research insights:

The difference in epitope recognition can be strategically leveraged in research designs. The Ab-108 antibody may be more suitable for detecting specific post-translational modifications near the Lys-108 site, while the Ab-20 antibody offers cross-species reactivity that enables comparative studies between human and mouse models . When investigating histone modifications in evolutionarily conserved pathways, researchers might consider using both antibodies to obtain a more comprehensive understanding of the protein's functional state.

What methodologies can be employed to study HIST1H2BC ubiquitylation patterns?

Studying HIST1H2BC ubiquitylation requires specialized methodologies to preserve and detect this post-translational modification. Based on established protocols, researchers can employ the following approach:

• Generate cell lines expressing FLAG-tagged H2B to facilitate detection of the ubiquitylated form

• Extract histones under denaturing conditions to preserve ubiquitylation:

  • Use buffer containing 1% SDS, 8M urea, 10mM Tris pH 8.0, 10mM EDTA

  • Include deubiquitylase inhibitors such as N-ethylmaleimide or ubiquitin aldehyde

• Resolve proteins on 15% SDS-polyacrylamide gels to achieve separation between unmodified and ubiquitylated forms

• Perform immunoblotting with anti-FLAG antibody to detect both unmodified and ubiquitylated forms based on molecular weight shift

• Verify specificity of the ubiquitylation signal using a lysine-to-arginine mutant (K119R in yeast or K120R in humans) as a negative control

This methodology has been successfully employed to demonstrate that histone H2B ubiquitylation is essential for proper gene regulation, including its role in gametogenesis and transcriptional processes .

How can HIST1H2BC (Ab-108) Antibody be utilized to investigate histone variant expression in disease models?

The HIST1H2BC (Ab-108) Antibody can be employed to investigate disease-specific alterations in histone variant expression, particularly in cancer and environmental exposure models. Research has shown that specific H2B variants exhibit altered expression patterns during cellular transformation processes such as epithelial-mesenchymal transition (EMT) .

For investigating histone variant changes in disease models, researchers can implement the following methodology:

• Establish appropriate disease models (e.g., inorganic arsenic-induced EMT)

• Extract histones using acid extraction methods to isolate the entire complement of histone proteins

• Employ Western blotting with HIST1H2BC (Ab-108) Antibody to detect expression level changes

• Complement antibody-based approaches with mass spectrometry:

  • Use electron capture dissociation-based top-down tandem mass spectrometry (MS/MS) for variant identification

  • Implement Fourier transform ion cyclotron resonance (FT-ICR) for high-resolution analysis

• Validate findings using quantitative reverse transcription real-time PCR to correlate protein expression with transcript levels

Studies using these approaches have revealed significant changes in H2B variant expression during carcinogenesis, specifically identifying increases in H2B1H/1K/1C/1J/1O and H2B2E/2F variants, and decreases in H2B1N/1D/1B variants during inorganic arsenic-mediated EMT .

What are the technical considerations for chromatin immunoprecipitation (ChIP) using HIST1H2BC (Ab-108) Antibody?

When performing ChIP with HIST1H2BC (Ab-108) Antibody to map genomic localization of this histone variant, several technical considerations should be addressed:

• Crosslinking optimization:

  • Standard 1% formaldehyde for 10 minutes at room temperature is typically sufficient

  • For studying interactions with non-coding RNAs or chromatin remodeling complexes, consider dual crosslinking with both formaldehyde and a protein-protein crosslinker like disuccinimidyl glutarate (DSG)

• Sonication parameters:

  • Optimize sonication conditions to generate DNA fragments of 200-500bp

  • Monitor fragmentation efficiency using agarose gel electrophoresis

• Antibody incubation:

  • Use 2-5μg of HIST1H2BC (Ab-108) Antibody per ChIP reaction

  • Dilute chromatin in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2mM EDTA, 16.7mM Tris-HCl pH 8.1, 167mM NaCl)

  • Incubate overnight at 4°C with rotation

• Controls:

  • Include isotype-matched IgG control

  • Consider using cells expressing FLAG-tagged H2B with anti-FLAG ChIP in parallel

  • For ubiquitylation studies, include H2B lysine mutants (K119R/K120R) as negative controls

• Data analysis:

  • For genome-wide studies, compare HIST1H2BC enrichment patterns with other histone marks

  • Particular attention should be paid to regions associated with gene regulation and chromatin organization

These technical considerations help ensure specific and reproducible ChIP results when using the HIST1H2BC (Ab-108) Antibody.

What are common issues encountered with HIST1H2BC (Ab-108) Antibody and how can they be resolved?

Researchers working with HIST1H2BC (Ab-108) Antibody may encounter several technical challenges. Here are common issues and their solutions:

• Low signal intensity in Western blots:

  • Increase antibody concentration within the recommended range (1:100-1:1000)

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

  • Optimize protein loading (20-50μg total protein recommended)

  • Ensure efficient transfer to membrane by using an appropriate transfer buffer and time

• High background in immunohistochemistry:

  • Increase blocking stringency (5% BSA in PBS-T instead of 3%)

  • Optimize antibody dilution (start with 1:50)

  • Include additional washing steps after primary and secondary antibody incubations

  • Consider using a different detection system with lower background

• Difficulties in detecting ubiquitylated forms:

  • Include deubiquitylase inhibitors in all buffers

  • Use fresh samples, as ubiquitylation can be lost during storage

  • Consider running control samples known to be enriched for H2B ubiquitylation

  • Use higher percentage gels (15-18%) to achieve better separation of unmodified and modified forms

• Cross-reactivity with other histone variants:

  • Validate specificity using recombinant histone variants

  • Consider pre-absorbing the antibody with recombinant proteins

  • Confirm results with mass spectrometry-based approaches to distinguish between highly similar variants

These troubleshooting approaches are based on established protocols and can significantly improve experimental outcomes when working with HIST1H2BC (Ab-108) Antibody.

How can HIST1H2BC (Ab-108) Antibody be used to study the functional relationship between histone modification and gene expression?

The HIST1H2BC (Ab-108) Antibody can be employed in integrative experimental approaches to elucidate the relationship between histone modification and gene expression:

• Combined ChIP-seq and RNA-seq analysis:

  • Perform ChIP-seq with HIST1H2BC (Ab-108) Antibody to map genomic localization

  • Conduct parallel RNA-seq to correlate histone variant localization with gene expression patterns

  • Analyze data to identify genes and pathways regulated by specific HIST1H2BC enrichment

• Sequential ChIP (Re-ChIP) for co-localization studies:

  • Perform first ChIP with HIST1H2BC (Ab-108) Antibody

  • Elute chromatin complexes and perform second ChIP with antibodies against specific modifications (e.g., H3K4me3, H3K79me3)

  • This approach can reveal whether HIST1H2BC co-localizes with specific activating or repressive marks

• Functional studies using genetic manipulation:

  • Implement CRISPR/Cas9-mediated gene editing to alter HIST1H2BC expression

  • Compare wild-type and mutant cells using ChIP-qPCR at specific genomic loci

  • Correlate changes in HIST1H2BC occupancy with alterations in gene expression

• Ubiquitylation-focused approaches:

  • Use HIST1H2BC (Ab-108) Antibody in combination with ubiquitylation studies

  • Employ genetic models with mutations in ubiquitylation machinery (e.g., Rtf1 mutations)

  • Analyze effects on non-coding RNA transcription and gene regulation

Research has demonstrated that histone H2B ubiquitylation plays crucial roles in various cellular processes, including gene expression regulation and noncoding RNA transcription . The HIST1H2BC (Ab-108) Antibody provides a valuable tool for investigating these relationships in different experimental contexts.

How does HIST1H2BC compare with other H2B variants in chromatin regulation?

HIST1H2BC is one of multiple H2B variants that exhibit high sequence conservation but can play distinct roles in chromatin regulation. Comparative analysis reveals:

Despite their high sequence conservation (differing by only a few amino acids), these variants can have distinct functional impacts on chromatin structure and gene expression . The HIST1H2BC (Ab-108) Antibody can be used to distinguish expression patterns of specific variants in different cellular contexts, though complementary mass spectrometry approaches may be necessary for comprehensive variant identification due to the high sequence similarity .

What is the relationship between HIST1H2BC ubiquitylation and other histone modifications?

HIST1H2BC ubiquitylation exists within a complex network of histone modifications that collectively regulate chromatin structure and gene expression. Research has revealed several key relationships:

• Cross-talk with H3 methylation:

  • H2B ubiquitylation is a prerequisite for H3K4 and H3K79 methylation

  • Mutations that prevent H2B ubiquitylation (such as K119R in yeast) impair these H3 methylation marks

  • This trans-histone pathway represents a key regulatory mechanism for gene expression

• Sequential addition and removal dynamics:

  • Both the addition and removal of ubiquitylation are required for proper gene regulation

  • Paradoxically, both preventing ubiquitylation (htb1 K119R mutation) and increasing ubiquitylation (ubp8 deletion) can lead to derepression of certain genes

  • This indicates a complex dynamic where the cycling of the modification, rather than its static presence, is functionally important

• Role in RNA polymerase II function:

  • H2B ubiquitylation influences RNA polymerase II occupancy and elongation

  • The modification affects chromatin dynamics during transcription, facilitating H2A-H2B dimer eviction and nucleosome reassembly

  • This process is particularly important for transcription through regions with complex chromatin structures

• Impact on non-coding RNA regulation:

  • H2B ubiquitylation plays a role in the termination of snoRNA transcripts

  • The modification also contributes to transcription at heterochromatic loci

  • These functions highlight its importance beyond traditional protein-coding gene regulation

Understanding these relationships provides insight into how HIST1H2BC and its modifications contribute to the broader epigenetic landscape and gene regulatory networks.

How can HIST1H2BC (Ab-108) Antibody be used in cancer research and biomarker development?

The HIST1H2BC (Ab-108) Antibody offers valuable applications in cancer research and biomarker development based on emerging evidence of H2B variant dysregulation in malignancy:

• Profiling histone variant expression in cancer progression:

  • Use the antibody to detect HIST1H2BC expression changes across cancer stages

  • Compare primary tumors with metastatic lesions to identify stage-specific alterations

  • Correlate expression patterns with clinical outcomes and treatment responses

• Investigating epithelial-mesenchymal transition (EMT):

  • Studies have demonstrated that specific H2B variants, including members of the H2B1C family, show altered expression during EMT

  • The antibody can be used to track these changes in experimental models of EMT and metastasis

  • This approach can reveal how histone variant switching contributes to cancer cell plasticity

• Environmental carcinogenesis research:

  • H2B variant expression changes have been observed in inorganic arsenic-mediated carcinogenesis

  • The antibody can help characterize histone variant profiles in response to environmental toxins

  • This application aids in understanding mechanisms of environmentally-induced cancers

• Biomarker development workflow:

  • Screen tissue microarrays from patient cohorts using immunohistochemistry with HIST1H2BC (Ab-108) Antibody

  • Validate findings using complementary techniques like mass spectrometry and qRT-PCR

  • Correlate histone variant profiles with existing cancer biomarkers and patient outcomes

  • Develop multiparameter biomarker panels incorporating histone variant expression

The clinical importance of histone variants is highlighted by reports identifying tissue-specific H2B variant regulation in specific cancer types, suggesting these variants may serve as valuable diagnostic or prognostic biomarkers .

What emerging technologies can enhance HIST1H2BC research beyond current antibody-based methods?

While the HIST1H2BC (Ab-108) Antibody provides valuable research capabilities, several emerging technologies offer complementary approaches for advancing histone variant research:

• CUT&RUN and CUT&Tag methodologies:

  • These techniques offer higher signal-to-noise ratios than traditional ChIP

  • They require significantly less starting material (thousands vs. millions of cells)

  • When adapted for use with HIST1H2BC antibodies, they could provide more sensitive genomic mapping

• CRISPR/Cas9 endogenous tagging:

  • Engineering endogenous HIST1H2BC with precision tags (FLAG, HA, etc.)

  • Enables live-cell imaging of histone dynamics using fluorescent tags

  • Provides a system for studying variant-specific functions without overexpression artifacts

• Single-cell epigenomics:

  • Adaptation of HIST1H2BC antibodies for single-cell CUT&Tag or similar approaches

  • Reveals cell-to-cell heterogeneity in histone variant distribution

  • Particularly valuable for studying complex tissues and tumors with heterogeneous cell populations

• Proteomics beyond antibodies:

  • Top-down MS/MS approaches for comprehensive histone variant profiling

  • Targeted proteomics using parallel reaction monitoring (PRM) for absolute quantification

  • Analysis of co-occurring post-translational modifications on specific histone variants

• Spatial transcriptomics/epigenomics:

  • Integration of histone variant profiling with spatial positioning in tissues

  • Reveals microenvironmental influences on histone variant expression

  • Particularly valuable for understanding heterogeneity in tumor samples

These emerging technologies, when combined with traditional antibody-based approaches, can provide unprecedented insights into the functions and regulation of HIST1H2BC and other histone variants.

What are the current limitations in understanding HIST1H2BC function and how might they be addressed?

Despite significant advances, several knowledge gaps remain in our understanding of HIST1H2BC function:

• Variant-specific functions:

  • Current challenge: Despite the identification of 22 H2B variant genes, specific functional roles for individual variants remain largely undefined

  • Research approach: Develop CRISPR-based selective knockout/knockin systems for individual variants followed by comprehensive phenotypic analysis

  • Expected outcome: Identification of tissue-specific or context-dependent functions of HIST1H2BC compared to other variants

• Regulatory mechanisms controlling variant expression:

  • Current challenge: The mechanisms governing differential expression of histone variants in various cellular contexts remain poorly understood

  • Research approach: Integrated analysis of transcription factor binding, chromatin accessibility, and post-transcriptional regulation at variant loci

  • Expected outcome: Elucidation of regulatory networks controlling histone variant switching during development and disease

• Post-translational modification patterns:

  • Current challenge: The full complement of modifications on specific variants and their combinatorial effects are not well characterized

  • Research approach: Advanced mass spectrometry technologies combined with genetic manipulation of modification enzymes

  • Expected outcome: Comprehensive modification maps of specific variants and their functional significance

• Chaperone-mediated deposition:

  • Current challenge: The mechanisms of variant-specific deposition into chromatin remain largely unexplored

  • Research approach: Proteomic identification of variant-specific chaperone interactions followed by functional validation

  • Expected outcome: Understanding how specific variants are targeted to particular genomic regions

Addressing these limitations requires interdisciplinary approaches combining genetic, biochemical, and computational methods. The development of more specific antibodies and complementary technologies will be crucial for advancing our understanding of HIST1H2BC and other histone variants in the coming years.