HIST1H2BC (Ab-108) Antibody

What is HIST1H2BC and what biological role does it play?

HIST1H2BC is a core histone protein belonging to the histone H2B family, specifically the histone cluster 1 H2bc variant. It functions as an essential structural component for packaging DNA into chromatin within the nucleus. HIST1H2BC plays a critical role in gene regulation, chromatin organization, and maintaining genome stability. The protein is involved in epigenetic regulation through post-translational modifications, particularly acetylation of lysine residues on its N-terminal tail . These modifications alter the chromatin structure and accessibility, thereby influencing transcriptional activity. HIST1H2BC has been implicated in various disease processes, including cancer and developmental disorders, where aberrant histone modifications can lead to dysregulated gene expression . Understanding the specific functions and modifications of HIST1H2BC provides valuable insights into fundamental cellular processes controlling gene expression and chromatin dynamics.

What are the key specifications of the HIST1H2BC (Ab-108) Antibody?

The HIST1H2BC (Ab-108) Antibody is a polyclonal antibody developed specifically against the region surrounding lysine 108 of human histone H2B type 1-C/E/F/G/I. The key specifications are summarized in the following table:

The antibody has been validated across multiple applications, including ELISA, Western blotting, immunohistochemistry, immunoprecipitation, and immunofluorescence . It specifically recognizes HIST1H2BC and related H2B variants, making it a versatile tool for epigenetic research studying histone modifications and chromatin structure.

What target cell types and samples have been validated with this antibody?

The HIST1H2BC (Ab-108) Antibody has been validated in several human cell lines and sample types, demonstrating consistent and reliable detection of the target protein. According to the product documentation, successful detection has been confirmed in:

• HeLa cells (cervical cancer cell line)

• 293 cells (embryonic kidney cell line)

• HepG2 cells (liver cancer cell line)

• HL60 cells (promyelocytic leukemia cell line)

• MCF-7 cells (breast cancer cell line)

For Western blot applications, whole cell lysates from these lines have shown positive detection of the target protein . For immunofluorescence applications, the antibody has been successfully used in HeLa cells with appropriate fixation and permeabilization protocols . This broad validation across multiple cell types suggests the antibody's reliability across different experimental systems, making it suitable for comparative studies of histone modifications in various cellular contexts.

What is the recommended protocol for using HIST1H2BC (Ab-108) in Western blotting experiments?

For optimal Western blotting results using the HIST1H2BC (Ab-108) Antibody, the following methodological approach is recommended:

• Sample Preparation:

  • Prepare whole cell lysates using a lysis buffer containing protease inhibitors

  • Include histone deacetylase inhibitors (such as sodium butyrate or TSA) to preserve acetylation marks

  • Quantify protein concentration using Bradford or BCA assay

• Gel Electrophoresis and Transfer:

  • Load 10-20 μg of protein per lane on a 15-18% SDS-PAGE gel (optimized for low molecular weight histones)

  • Use a PVDF membrane for transfer (preferred over nitrocellulose for histone proteins)

  • Perform transfer at 100V for 1 hour in cold transfer buffer with 20% methanol

• Antibody Incubation:

  • Block membrane with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature

  • Dilute HIST1H2BC (Ab-108) Antibody at 1:100-1:1000 in blocking buffer

  • Incubate overnight at 4°C with gentle agitation

  • Wash 3 times with TBST, 5 minutes each

  • Incubate with HRP-conjugated anti-rabbit secondary antibody (1:5000) for 1 hour at room temperature

  • Wash 3 times with TBST, 5 minutes each

• Detection and Controls:

  • Develop using enhanced chemiluminescence (ECL) substrate

  • Expected band size for H2B is approximately 14 kDa

  • Include positive controls such as HeLa or 293 whole cell lysates

  • Consider including a loading control targeting total histone H3 or H4

This protocol has been optimized for detecting HIST1H2BC in human samples and may require adjustment based on specific experimental conditions and cell types.

How should HIST1H2BC (Ab-108) be used for immunofluorescence staining?

For effective immunofluorescence (IF) staining using the HIST1H2BC (Ab-108) Antibody, follow this detailed protocol:

• Cell Preparation:

  • Grow cells on sterile coverslips in appropriate culture medium

  • Consider treating with histone deacetylase inhibitors to enhance acetylation signals if studying acetylated histones

• Fixation and Permeabilization:

  • Fix cells in 4% formaldehyde (paraformaldehyde) for 15 minutes at room temperature

  • Permeabilize with 0.1% Triton X-100 in PBS for 10 minutes

  • Wash 3 times with PBS, 5 minutes each

• Blocking and Antibody Incubation:

  • Block with 1-5% BSA in PBS for 30-60 minutes at room temperature

  • Dilute HIST1H2BC (Ab-108) Antibody at 1:1-1:10 in blocking solution

  • Incubate overnight at 4°C in a humid chamber

  • Wash 3 times with PBS, 5 minutes each

  • Incubate with fluorophore-conjugated anti-rabbit secondary antibody (1:200-1:500) for 1 hour at room temperature in the dark

  • Wash 3 times with PBS, 5 minutes each

• Counterstaining and Mounting:

  • Counterstain nuclei with DAPI (1 μg/ml) for 5 minutes

  • Mount coverslips on slides using anti-fade mounting medium

  • Seal edges with nail polish and store at 4°C protected from light

• Controls and Imaging:

  • Include a negative control (secondary antibody only)

  • Compare staining pattern with known nuclear markers

  • Use confocal microscopy for optimal resolution of nuclear staining patterns

  • Expect predominantly nuclear localization with potential enrichment at specific chromatin regions

This protocol has been validated in HeLa cells and should produce clear nuclear staining corresponding to HIST1H2BC localization within chromatin .

How can HIST1H2BC (Ab-108) be utilized for chromatin immunoprecipitation (ChIP) experiments?

While not explicitly mentioned in the search results, the HIST1H2BC (Ab-108) Antibody can be adapted for ChIP experiments based on its applications in immunoprecipitation. Here is a methodological approach for ChIP:

• Crosslinking and Chromatin Preparation:

  • Crosslink cells with 1% formaldehyde for 10 minutes at room temperature

  • Quench with 125 mM glycine for 5 minutes

  • Lyse cells in appropriate buffers and sonicate to generate DNA fragments of 200-500 bp

  • Reserve 10% of sonicated chromatin as input control

• Immunoprecipitation:

  • Pre-clear chromatin with protein A/G beads for 1 hour at 4°C

  • Remove beads and add HIST1H2BC (Ab-108) Antibody at 1:200-1:500 dilution

  • Incubate overnight at 4°C with rotation

  • Add protein A/G beads and incubate for 2-3 hours at 4°C

  • Wash beads with increasingly stringent wash buffers

• DNA Recovery and Analysis:

  • Reverse crosslinks by heating at 65°C overnight

  • Treat with RNase A and Proteinase K

  • Purify DNA using phenol-chloroform extraction or commercial kits

  • Analyze by qPCR, library preparation for sequencing, or other downstream applications

• Controls and Validation:

  • Include IgG negative control

  • Include a positive control antibody targeting well-characterized histone marks

  • Validate enrichment at known regions associated with H2B modifications

  • Compare with H3K27ac enrichment patterns when studying enhancer regions

This protocol should be optimized for each cell type and experimental system, with special attention to sonication conditions and antibody concentration.

How does H2B acetylation compare to H3K27ac in marking active enhancers?

Recent research has revealed distinctive roles for histone H2B N-terminus multisite lysine acetylation (H2BNTac) compared to the well-established H3K27ac mark in identifying active enhancers. The comparison reveals several important distinctions:

• Specificity in Marking Regulatory Elements:

  • H2BNTac prominently marks candidate active enhancers and a subset of promoters

  • H2BNTac distinctively discriminates active enhancers from ubiquitously active promoters

  • Unlike H3K27ac, which marks both enhancers and active promoters broadly, H2BNTac shows higher specificity for enhancers

• Mechanistic Differences:

  • H2BNTac is specifically catalyzed by CBP/p300, whereas H3K27ac can be catalyzed by multiple histone acetyltransferases

  • H2A-H2B dimers (containing H2BNTac) are more rapidly exchanged through transcription-induced nucleosome remodeling compared to H3-H4 tetramers (containing H3K27ac)

• Predictive Power:

  • H2BNTac intensity more accurately predicts enhancer strength compared to other histone marks

  • H2BNTac outperforms current state-of-the-art models in predicting CBP/p300 target genes

  • H2BNTac-positive candidate enhancers show a high validation rate in orthogonal enhancer activity assays

• Regulatory Dynamics:

  • A-485 (a CBP/p300 inhibitor) reduces H2BNTac more strongly in promoters and actively transcribed gene body regions than in distal regions

  • H2BNTac is regulated by histone deacetylases (HDACs) 1 and 2, and its levels increase with class I deacetylase inhibitors

These findings suggest that using HIST1H2BC (Ab-108) Antibody to detect H2B acetylation can provide complementary and potentially more specific information about enhancer activity compared to standard H3K27ac profiling. Integrating both marks in epigenomic studies offers a more comprehensive understanding of active regulatory elements in the genome.

What technical considerations are important when interpreting HIST1H2BC (Ab-108) results across different experimental systems?

When interpreting results obtained with HIST1H2BC (Ab-108) Antibody across different experimental platforms, several technical considerations should be taken into account:

• Epitope Accessibility in Different Applications:

  • In Western blot applications, the denatured protein presents the epitope differently than in fixed cells (IHC/IF) or native conformation (IP)

  • The lysine 108 region may have differential accessibility depending on chromatin compaction state and nuclear architecture

  • Consider using multiple detection methods to confirm findings

• Cross-Reactivity Considerations:

  • The antibody targets a region of H2B that is highly conserved across multiple H2B variants (H2BC4, H2BC6, H2BC7, H2BC8, H2BC10)

  • Results may reflect combined signal from multiple H2B variants rather than specifically HIST1H2BC

  • Supplementary validation with variant-specific methods may be required for absolute specificity

• Influence of Post-Translational Modifications:

  • Acetylation or other modifications near the antibody binding site may affect epitope recognition

  • Consider the impact of histone deacetylase inhibitors or other epigenetic modulators on binding efficiency

  • When studying specific modifications, confirm with modification-specific antibodies

• Cell Type and Physiological State Variations:

  • Expression and modification patterns of histones vary across cell types and physiological states

  • Chromatin accessibility differences between cell types may affect antibody penetration in IF/IHC

  • Establish baseline detection levels specific to each experimental system

• Quantitative Analysis Considerations:

  • Signal intensity may not linearly correlate with protein abundance due to epitope masking effects

  • For ChIP-seq applications, normalize signals appropriately and consider sequencing depth variations

  • When comparing H2B modifications with other histone marks (e.g., H3K27ac), account for different antibody efficiencies

These technical considerations should guide experimental design, data interpretation, and troubleshooting when working with the HIST1H2BC (Ab-108) Antibody across different research applications.

How can specificity and potential cross-reactivity of HIST1H2BC (Ab-108) be assessed?

Assessing the specificity and potential cross-reactivity of HIST1H2BC (Ab-108) Antibody is crucial for accurate data interpretation. Here's a methodological approach:

• Peptide Competition Assay:

  • Pre-incubate the antibody with excess immunizing peptide (sequence around Lys-108)

  • Perform parallel experiments with blocked and unblocked antibody

  • Significant signal reduction in the blocked condition confirms specificity for the target epitope

• Genetic Validation:

  • Use CRISPR/Cas9 to knockout or knockdown HIST1H2BC

  • Compare antibody signal between wild-type and knockout/knockdown samples

  • Loss of signal in genetic models strongly supports specificity

• Cross-Reactivity Testing:

  • Test the antibody against recombinant proteins of various H2B variants

  • Perform dot blots or Western blots with purified histones

  • Quantify relative binding to different H2B variants and non-H2B histones

• Modification-Specific Validation:

  • If studying acetylation or other modifications, compare antibody recognition in samples treated with or without HDAC inhibitors

  • Use mass spectrometry to confirm the presence of specific modifications at the target site

  • Compare with established modification-specific antibodies targeting the same region

• Species Cross-Reactivity Assessment:

  • Test the antibody on samples from different species with varying degrees of sequence homology

  • Although primarily reactive with human samples, conservation of histone sequences may permit cross-species applications

  • Align sequences across species to predict potential cross-reactivity

These validation approaches should be implemented systematically to establish confidence in experimental results obtained with the HIST1H2BC (Ab-108) Antibody.

What are optimal sample preparation methods to preserve histone modifications when using HIST1H2BC (Ab-108)?

To maintain the integrity of histone modifications when working with HIST1H2BC (Ab-108) Antibody, the following sample preparation methods are recommended:

• Cell and Tissue Harvesting:

  • Minimize the time between harvesting and fixation/extraction

  • Process samples on ice whenever possible

  • Consider flash-freezing tissues if immediate processing is not possible

• Histone Extraction Methods:

  • For Western blotting or IP applications, use acid extraction methods:

    • Lyse cells in Triton Extraction Buffer (PBS with 0.5% Triton X-100, 2mM PMSF, 0.02% NaN₃)

    • Extract histones with 0.2N HCl overnight at 4°C

    • Neutralize with 1M Tris base and quantify protein concentration

  • Include deacetylase inhibitors (5-10mM sodium butyrate, 1μM TSA, or commercial cocktails)

  • Add protease inhibitors to prevent degradation

• Fixation for Immunohistochemistry/Immunofluorescence:

  • Use freshly prepared 4% paraformaldehyde for optimal epitope preservation

  • Limit fixation time to 15-20 minutes at room temperature

  • For tissues, consider using PAXgene fixation or other methods that better preserve protein modifications

  • Perform antigen retrieval using citrate buffer (pH 6.0) for formalin-fixed samples

• Chromatin Preparation for ChIP:

  • Use dual crosslinking (1.5mM EGS followed by 1% formaldehyde) for improved histone modification preservation

  • Include both protease and phosphatase inhibitors in all buffers

  • Add deacetylase inhibitors to maintain acetylation marks

  • Optimize sonication conditions to ensure efficient chromatin fragmentation while preserving epitopes

• Storage Considerations:

  • Store extracted histones at -80°C with glycerol as a cryoprotectant

  • Avoid repeated freeze-thaw cycles

  • For long-term storage of fixed cells/tissues, consider keeping samples in PBS with 0.02% sodium azide at 4°C rather than freezing

These methodological considerations are essential for maintaining the native state of histone modifications and ensuring reliable detection with the HIST1H2BC (Ab-108) Antibody.

How can HIST1H2BC (Ab-108) be combined with other histone mark antibodies for comprehensive epigenetic profiling?

For comprehensive epigenetic profiling, HIST1H2BC (Ab-108) Antibody can be strategically combined with other histone mark antibodies through several methodological approaches:

• Sequential ChIP (Re-ChIP) Strategy:

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

  • Elute chromatin complexes under mild conditions

  • Perform second round of ChIP with antibodies against other marks (H3K27ac, H3K4me1, H3K4me3)

  • This approach identifies genomic regions with co-occurrence of H2B modifications and other histone marks

  • Particularly valuable for studying bivalent domains or complex regulatory elements

• Multimodal Imaging Approaches:

  • Perform multiplexed immunofluorescence with HIST1H2BC (Ab-108) and antibodies against:

    • Other histone marks (H3K27ac, H3K9me3, H3K27me3)

    • Chromatin remodeling factors (CBP/p300)

    • Transcription factors of interest

  • Use secondary antibodies with spectrally distinct fluorophores

  • Analyze co-localization patterns to infer functional interactions

• Integrated ChIP-seq Analysis Framework:

  • Perform parallel ChIP-seq experiments with HIST1H2BC (Ab-108) and other histone mark antibodies

  • Integrate datasets using computational approaches:

    • Peak overlap analysis to identify regions with multiple marks

    • Correlation analysis of signal intensities across the genome

    • Chromatin state modeling (e.g., ChromHMM) including H2BNTac data

  • This approach has revealed that H2BNTac and H3K27ac show different enrichment patterns at enhancers versus promoters

• Correlative Multi-Omics Integration:

  • Combine ChIP-seq data using HIST1H2BC (Ab-108) with:

    • ATAC-seq for chromatin accessibility

    • RNA-seq for gene expression correlation

    • GRO-seq for nascent transcription

  • This integrative approach can reveal how H2B modifications relate to transcriptional activity and chromatin structure

  • Particularly valuable for understanding enhancer-promoter interactions

• Mass Spectrometry Validation:

  • Perform immunoprecipitation with HIST1H2BC (Ab-108)

  • Analyze precipitated histones by mass spectrometry

  • Identify co-occurring modifications on the same histone molecules

  • This approach provides direct evidence of modification crosstalk not obtainable by ChIP alone

These integrated approaches leverage the strengths of HIST1H2BC (Ab-108) Antibody while providing a more comprehensive view of the epigenetic landscape, particularly in enhancer regions where H2BNTac has shown distinct regulatory patterns.

What experimental designs are recommended for studying the role of HIST1H2BC acetylation in disease models?

When investigating the role of HIST1H2BC acetylation in disease models using the HIST1H2BC (Ab-108) Antibody, the following experimental design frameworks are recommended:

• Comparative Profiling in Disease vs. Normal States:

  • Compare H2B acetylation patterns between:

    • Tumor tissue and matched normal tissue

    • Disease model cell lines and non-disease controls

    • Patient-derived samples with different disease stages

  • Use multiple detection methods (Western blot, ChIP-seq, IF) for comprehensive assessment

  • Correlate findings with disease progression markers

  • Given the role of H2BNTac in marking active enhancers , focus analysis on disease-specific enhancer regions

• Pharmacological Modulation Studies:

  • Treat disease models with epigenetic modulators targeting relevant pathways:

    • CBP/p300 inhibitors like A-485, which specifically affects H2BNTac

    • HDAC inhibitors that increase H2BNTac levels

    • Disease-specific therapeutic compounds

  • Monitor changes in HIST1H2BC acetylation patterns

  • Correlate with phenotypic changes and transcriptional responses

  • Design time-course experiments to capture dynamic regulation

• Genetic Perturbation Approaches:

  • Engineer cellular models with:

    • CRISPR/Cas9 targeting of CBP/p300

    • CRISPR activation/inhibition of HIST1H2BC

    • Site-specific mutation of acetylation sites (e.g., K108)

  • Analyze consequences on enhancer function and gene expression

  • Rescue experiments to confirm specificity of observed effects

  • Compare with disease-specific genomic alterations

• Enhancer-Focused Functional Analysis:

  • Identify disease-relevant enhancers marked by H2BNTac

  • Validate enhancer function using reporter assays

  • Perform targeted epigenome editing of these regions

  • Link enhancer activity to target gene expression

  • This approach leverages H2BNTac's role in marking active enhancers and predicting enhancer strength

• Multi-Omics Integration:

  • Combine H2BNTac profiling with:

    • Gene expression analysis (RNA-seq)

    • Chromatin accessibility (ATAC-seq)

    • DNA methylation profiles

    • Single-cell approaches for heterogeneity assessment

  • Integrate with patient clinical data when available

  • Apply machine learning approaches to identify patterns

  • Develop predictive models for disease progression or therapeutic response

These experimental frameworks provide comprehensive approaches to understand the role of HIST1H2BC acetylation in disease contexts, with particular emphasis on enhancer regulation where H2BNTac has demonstrated significant biological relevance.