HIST1H2BC (Ab-23) Antibody

What is HIST1H2BC and why is it important in epigenetic research?

HIST1H2BC (Histone H2B type 1-C/E/F/G/I) is a core component of nucleosomes, which are the fundamental units of chromatin structure. This protein plays a critical role in chromatin remodeling and transcriptional regulation, making it essential for processes including cell differentiation, development, and disease progression .

HIST1H2BC functions by wrapping and compacting DNA into chromatin, which limits DNA accessibility to cellular machinery. This regulatory function makes it a central player in transcription regulation, DNA repair, DNA replication, and chromosomal stability . DNA accessibility is regulated through post-translational modifications of histones (including HIST1H2BC), forming part of the "histone code" that governs gene expression.

Researchers studying epigenetics, gene regulation, and chromatin biology find HIST1H2BC antibodies essential for understanding the function and regulation of this important histone protein in various experimental settings .

What are the main applications and validation parameters for HIST1H2BC (Ab-23) Antibody?

HIST1H2BC (Ab-23) Antibody has been validated for multiple research applications, each with specific optimization parameters:

For IHC applications, the antibody has been specifically tested on paraffin-embedded human tissues using a Leica BondTM system. After dewaxing and hydration, high-pressure antigen retrieval is performed in citrate buffer (pH 6.0), followed by blocking with 10% normal goat serum for 30 minutes at room temperature .

How does HIST1H2BC (Ab-23) Antibody compare to other histone H2B antibodies?

HIST1H2BC (Ab-23) Antibody differs from other histone H2B antibodies in several key aspects:

Target Specificity:

• HIST1H2BC (Ab-23) Antibody specifically targets the region around lysine 23 (Lys-23) of human HIST1H2BC protein

• Other H2B antibodies may target different epitopes, modifications, or variants of H2B

Host and Format:

• HIST1H2BC (Ab-23) Antibody is a rabbit polyclonal antibody

• Some alternatives like anti-Histone H2B antibody (ab52484) are mouse monoclonal antibodies

Species Reactivity:

• HIST1H2BC (Ab-23) Antibody primarily reacts with human samples

• Alternative H2B antibodies like ab52484 show broader reactivity (human, mouse, rat)

Applications:

• While HIST1H2BC (Ab-23) Antibody is validated for ELISA, WB, IHC, and IF , other antibodies may have additional validations for applications such as flow cytometry

When selecting between different H2B antibodies, researchers should consider the specific requirements of their experimental system, including target epitope, species, and required applications.

What is the optimal protocol for using HIST1H2BC (Ab-23) Antibody in Western blot applications?

For optimal Western blot results with HIST1H2BC (Ab-23) Antibody, follow this methodological protocol:

Sample Preparation:

• Prepare whole cell lysates or nuclear extracts (recommended for histone proteins)

• Use protein extraction buffers containing protease inhibitors to prevent degradation

Gel Electrophoresis:

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

• Include positive control samples (human cell line lysates)

• Run gel according to standard protocols

Transfer and Blocking:

• Transfer proteins to PVDF or nitrocellulose membrane

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

Antibody Incubation:

• Dilute HIST1H2BC (Ab-23) Antibody 1:100-1:1000 in blocking buffer

• Incubate overnight at 4°C with gentle agitation

• Wash membrane 3-5 times with TBST, 5 minutes each

• Incubate with HRP-conjugated anti-rabbit secondary antibody at 1:5000 dilution

• Wash thoroughly 3-5 times with TBST

Detection:

• Develop using ECL substrate and appropriate imaging system

• Expected band size: 14 kDa (HIST1H2BC's predicted molecular weight)

Recommendations for Troubleshooting:

• If background is high, increase blocking time or wash stringency

• If signal is weak, decrease antibody dilution or increase protein loading

• Include controls to confirm specificity (isotype control, blocking peptide)

How should antigen retrieval be optimized for HIST1H2BC (Ab-23) Antibody in immunohistochemistry?

Optimizing antigen retrieval is crucial for successful IHC with HIST1H2BC (Ab-23) Antibody. Follow this methodological approach:

Fixation Considerations:

• Use 10% neutral buffered formalin for tissue fixation

• Avoid overfixation, which can mask epitopes

Recommended Antigen Retrieval Protocol:

• Deparaffinize sections in xylene and rehydrate through graded alcohols to water

• Perform heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) under high pressure

• Alternative: Try EDTA buffer (pH 8.0) if citrate buffer yields insufficient results

• Heat at high pressure for optimal epitope exposure

• Cool sections to room temperature gradually

• Rinse thoroughly in PBS or TBS

Staining Protocol:

• Block endogenous peroxidase activity with 3% H₂O₂

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

• Dilute HIST1H2BC (Ab-23) Antibody 1:10-1:100 in 1% BSA

• Incubate overnight at 4°C in a humidified chamber

• Wash thoroughly with buffer

• Apply biotinylated secondary antibody followed by HRP-conjugated detection system

• Develop with DAB substrate and counterstain with hematoxylin

Validation Strategy:

• Test multiple antibody dilutions (1:10, 1:50, 1:100)

• Compare different antigen retrieval conditions

• Use positive control tissues (human glioma or ovarian cancer samples have shown good results)

• Include a negative control by omitting primary antibody

Successful implementation of this protocol has been demonstrated in paraffin-embedded human glioma performed on a Leica BondTM system .

What controls are essential when using HIST1H2BC (Ab-23) Antibody for chromatin immunoprecipitation (ChIP)?

For reliable ChIP experiments with HIST1H2BC (Ab-23) Antibody, the following controls are essential:

Input Control:

• Reserve 5-10% of chromatin before immunoprecipitation

• This represents the starting material and allows quantification of enrichment

Negative Control Antibody:

• Use a non-specific IgG from the same species (rabbit IgG)

• Process identically to experimental samples

• Establishes background enrichment levels

Positive Control Target:

No Antibody Control:

• Process a sample with beads but no antibody

• Identifies non-specific binding to beads

Technical Considerations:

• Chromatin Preparation:

  • Properly fix cells with formaldehyde (typically 10 minutes)

  • Aim for DNA fragments of 200-500 bp

• Antibody Amount:

  • Use 2-5 μg of HIST1H2BC (Ab-23) Antibody per ChIP reaction

• Quantification:

  • Use real-time PCR (Sybr green approach) for quantifying immunoprecipitated DNA

• Data Analysis:

  • Calculate enrichment relative to input and IgG control

  • Present data as percent input or fold enrichment over IgG

When selecting targets for analysis, consider the chromatin landscape of your experimental system and include known H2B-enriched regions as positive controls. The ChIP validated antibodies collection provides useful reference for additional controls and comparative analysis .

How does HIST1H2BC contribute to chromatin remodeling and gene expression regulation?

HIST1H2BC plays several crucial roles in chromatin dynamics and gene regulation through multiple mechanisms:

Nucleosome Structure and Organization:

• HIST1H2BC forms part of the histone octamer core around which DNA wraps to form nucleosomes

• Each nucleosome consists of ~146 bp of DNA wrapped around a histone octamer containing two copies each of H2A, H2B, H3, and H4

• HIST1H2BC pairs with another H2B molecule and interacts with H2A dimers to form the nucleosome structure

Chromatin Accessibility Regulation:

• By compacting DNA, HIST1H2BC limits accessibility to transcription factors and other regulatory proteins

• This compaction is dynamic and regulated through various mechanisms

Post-translational Modifications (PTMs):

• HIST1H2BC undergoes various modifications including:

  • Acetylation (particularly at lysine residues including K23)

  • Ubiquitination (notably at K120)

  • Phosphorylation (during mitosis and DNA damage response)

  • Methylation

• These modifications form part of the "histone code" that regulates chromatin structure and function

Functional Impact on Cellular Processes:

• Gene expression: Modified HIST1H2BC can recruit chromatin-modifying complexes that promote or repress transcription

• DNA repair: HIST1H2BC participates in the DNA damage response pathway

• DNA replication: Nucleosome disassembly and reassembly during replication involves HIST1H2BC

• Cell differentiation: Changes in HIST1H2BC modifications accompany developmental processes

• Disease development: Altered HIST1H2BC regulation is implicated in cancer and other diseases

Research using HIST1H2BC (Ab-23) Antibody can help elucidate these roles by mapping HIST1H2BC distribution and modifications across the genome in different cellular contexts.

What are the potential cross-reactivity issues with HIST1H2BC (Ab-23) Antibody and how can they be mitigated?

Sources of Cross-Reactivity:

• Sequence Homology: High conservation among histone variants may lead to cross-reactivity with other H2B variants

• Post-translational Modifications: Modifications near lysine 23 may affect antibody binding

• Non-specific Binding: The polyclonal nature of the antibody may result in some non-specific interactions

Mitigation Strategies:

Application-Specific Considerations:

• For IHC: Include adjacent sections with isotype control antibody

• For WB: Verify single band of expected molecular weight (14 kDa)

• For IF: Compare nuclear localization pattern with known H2B distribution

• For ChIP: Include IgG control to establish background levels

Implementing these strategies will help ensure the specificity of your results when using HIST1H2BC (Ab-23) Antibody across different experimental applications.

How does post-translational modification of HIST1H2BC affect antibody recognition?

Post-translational modifications (PTMs) of HIST1H2BC can significantly impact antibody recognition, particularly for antibodies targeting specific residues like HIST1H2BC (Ab-23) Antibody:

Effect of PTMs on Antibody Binding:

• Direct Epitope Modification:

  • Since HIST1H2BC (Ab-23) Antibody targets the region around lysine 23 , modifications at this site (such as acetylation, methylation, or ubiquitination) would likely interfere with antibody binding

  • This creates both a limitation and potential application depending on research goals

• Conformational Changes:

  • PTMs near but not directly at the epitope can alter protein conformation

  • These conformational changes may enhance or inhibit antibody access to the target site

• Protein-Protein Interactions:

  • Modified histones often recruit binding partners

  • These interactions may mask the epitope and prevent antibody binding

Common HIST1H2BC Modifications and Their Impact:

Methodological Approaches:

• For Studies of Total HIST1H2BC Regardless of Modification:

  • Select antibodies targeting regions less frequently modified

  • Consider antibodies recognizing the C-terminal region

• For Studies of Specific Modified Forms:

  • Use modification-specific antibodies (e.g., anti-acetyl-H2B(K23))

  • Complement with mass spectrometry analysis for comprehensive PTM profiling

• For Comprehensive Analysis:

  • Use multiple antibodies targeting different epitopes and specific modifications

  • Correlate results across multiple techniques (ChIP, WB, IF)

Understanding these interactions is crucial for proper experimental design and interpretation of results when using HIST1H2BC (Ab-23) Antibody in research contexts where histone modifications play important roles.

What is the role of HIST1H2BC in cancer biology and how can HIST1H2BC (Ab-23) Antibody contribute to cancer research?

HIST1H2BC has emerging roles in cancer biology through several mechanisms, and HIST1H2BC (Ab-23) Antibody provides valuable research tools for investigating these connections:

HIST1H2BC in Cancer Pathobiology:

• Epigenetic Dysregulation:

  • Altered histone modifications affect gene expression patterns in cancer cells

  • HIST1H2BC modifications contribute to aberrant activation or silencing of cancer-related genes

• Chromatin Structure Changes:

  • Cancer cells often display altered chromatin organization

  • As a core nucleosomal protein, HIST1H2BC mediates these structural changes

• DNA Damage Response:

  • HIST1H2BC participates in DNA repair mechanisms

  • Defects in these pathways contribute to genomic instability in cancer

• Antimicrobial Properties:

  • HIST1H2BC has broad antibacterial activity and may contribute to antimicrobial barriers in epithelial tissues

  • This function might affect tumor microenvironment interactions

Research Applications of HIST1H2BC (Ab-23) Antibody in Cancer Research:

Specific Cancer Types with Validated Applications:

• Human glioma tissues have shown positive HIST1H2BC staining with the antibody

• Ovarian cancer tissues have also been successfully used for validation

Research Strategy Recommendations:

• Use HIST1H2BC (Ab-23) Antibody in combination with markers of cell proliferation, apoptosis, or other cancer hallmarks

• Apply the antibody across cancer progression models to track changes in HIST1H2BC expression or modification

• Combine with next-generation sequencing approaches to correlate HIST1H2BC binding with transcriptional changes

By utilizing HIST1H2BC (Ab-23) Antibody in these research contexts, investigators can advance understanding of epigenetic mechanisms in cancer and potentially identify new therapeutic targets.