Acetyl-HIST1H2BC (K120) Antibody

What is the Acetyl-HIST1H2BC (K120) antibody and what epitope does it recognize?

The Acetyl-HIST1H2BC (K120) antibody is a polyclonal antibody raised in rabbits that specifically recognizes the acetylation modification at lysine 120 (K120) of the Histone H2B type 1-C/E/F/G/I protein. The immunogen used for antibody production is typically a synthetic peptide sequence around the site of acetylated lysine 120 derived from human Histone H2B type 1-C/E/F/G/I . This antibody enables researchers to specifically detect and study this post-translational modification and its role in gene regulation and chromatin structure.

What is the biological significance of histone H2B acetylation?

Histone H2B acetylation, including at K120, is a critical post-translational modification involved in the epigenetic regulation of gene expression. Core components of nucleosomes, including H2B, wrap and compact DNA into chromatin, limiting DNA accessibility to cellular machineries that require DNA as a template . Acetylation of lysine residues on histones generally neutralizes their positive charge, weakening histone-DNA interactions and creating a more accessible chromatin structure. This modification plays central roles in transcription regulation, DNA repair, DNA replication, and chromosomal stability . The specific acetylation at K120 is associated with active gene transcription and is often found in promoter and enhancer regions of actively transcribed genes.

How does the Acetyl-HIST1H2BC (K120) antibody differ from other histone modification antibodies?

The Acetyl-HIST1H2BC (K120) antibody specifically targets the acetylation modification at lysine 120 of histone H2B, distinguishing it from antibodies that recognize other modifications. For comparison, the Acetyl-HIST1H2BC (K12) antibody recognizes acetylation at lysine 12 , while the 2-hydroxyisobutyryl-HIST1H2BC (K120) antibody detects a different modification (2-hydroxyisobutyrylation) at the same K120 position . Each modification-specific antibody enables researchers to study distinct epigenetic marks that may have different functional consequences. The specificity of these antibodies is typically validated using peptide arrays or dot blot analyses with modified and unmodified peptides to ensure they selectively recognize the intended modification at the precise lysine residue.

What are the validated applications for Acetyl-HIST1H2BC (K120) antibody in epigenetic research?

The Acetyl-HIST1H2BC (K120) antibody has been validated for several research applications:

These applications allow researchers to study the presence, distribution, and dynamics of H2B K120 acetylation in various experimental contexts . When designing experiments, it's recommended to optimize antibody concentrations for each specific application and cell/tissue type.

How should ChIP experiments be designed and optimized when using histone acetylation antibodies?

When designing ChIP experiments with histone acetylation antibodies like Acetyl-HIST1H2BC (K120), several methodological considerations are important:

• Chromatin preparation: Use freshly prepared cross-linked chromatin sheared to 200-500 bp fragments. For histone modifications, formaldehyde fixation for 10 minutes at room temperature is typically sufficient .

• Antibody titration: Perform an antibody titration (0.5-5 μg per ChIP experiment) to determine optimal concentration, as demonstrated with similar histone antibodies where 0.5, 1, 2, and 5 μg antibody amounts were tested per ChIP experiment .

• Controls: Include appropriate controls such as IgG (negative control) and primers for known positive regions (e.g., active promoters like GAPDH and ACTB) and negative regions (e.g., inactive genes like MYOD1 or heterochromatic regions like Sat2) .

• Quantification: Express recovery as a percentage of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis) .

• Validation: Consider performing ChIP-seq to obtain genome-wide distribution patterns, which can validate the antibody's specificity and provide comprehensive insights into the modification's distribution .

For optimal results, chromatin from approximately 1.5 million cells is recommended based on protocols used with similar histone modification antibodies .

What are the key considerations for Western blot analysis using the Acetyl-HIST1H2BC (K120) antibody?

For successful Western blot analysis with Acetyl-HIST1H2BC (K120) antibody:

• Sample preparation: Use histone extracts (15 μg) or whole cell extracts (25 μg), with histone extracts providing cleaner results for histone-specific modifications .

• Gel selection: Use high percentage (15-18%) SDS-PAGE gels to properly resolve the low molecular weight histone proteins (Histone H2B has a predicted band size of 14 kDa) .

• Transfer conditions: Optimize protein transfer using PVDF membranes and proper transfer conditions for small proteins.

• Blocking: Block with 5% non-fat dry milk or 5% BSA in TBST to minimize background.

• Antibody dilution: Use recommended dilutions (1:100-1:1000) for primary antibody incubation . For similar histone antibodies, 1:1000 dilution has shown good results .

• Controls: Include recombinant histones (H2A, H2B, H3, H4) as controls to verify specificity, and consider using sodium butyrate-treated samples (a histone deacetylase inhibitor) as positive controls for acetylation marks .

• Detection: Use appropriate secondary antibodies and sensitive chemiluminescent detection systems.

When interpreting results, be aware that the antibody should detect a band at approximately 14 kDa corresponding to histone H2B .

How can Acetyl-HIST1H2BC (K120) antibody be used to investigate the interplay between different histone modifications?

Investigating the interplay between histone modifications requires sophisticated experimental approaches:

• Sequential ChIP (Re-ChIP): This technique involves performing successive immunoprecipitations with different modification-specific antibodies. For example, performing ChIP first with Acetyl-HIST1H2BC (K120) antibody followed by ChIP with antibodies against other modifications can identify genomic regions where multiple modifications co-occur.

• Mass spectrometry validation: Combined with immunoprecipitation, mass spectrometry can identify peptides with multiple modifications, allowing researchers to confirm co-occurrence of K120 acetylation with other modifications on the same histone tail.

• Combinatorial analysis: ChIP-seq data from Acetyl-HIST1H2BC (K120) can be computationally integrated with datasets for other histone marks to identify correlation patterns and potential functional relationships between different modifications.

• Genetic manipulation: Using CRISPR/Cas9 to generate lysine-to-arginine mutations at specific sites can help determine how preventing acetylation at one site affects modifications at other sites.

• Inhibitor studies: Treating cells with specific histone acetyltransferase (HAT) or histone deacetylase (HDAC) inhibitors can reveal how changes in acetylation at K120 affect other histone modifications.

These approaches can help elucidate the "histone code" and understand how combinations of modifications regulate chromatin structure and function .

What are the best strategies for troubleshooting non-specific binding or high background when using histone acetylation antibodies?

When encountering non-specific binding or high background with Acetyl-HIST1H2BC (K120) or other histone acetylation antibodies, consider these troubleshooting strategies:

• Validate antibody specificity: Perform dot blot analysis with modified and unmodified peptides to confirm specificity. Similar histone modification antibodies have been validated using dot blots with peptide concentrations ranging from 0.2-100 pmol to demonstrate specificity .

• Optimize blocking conditions: Test different blocking reagents (BSA, non-fat milk, commercial blockers) and concentrations to reduce non-specific binding.

• Increase washing stringency: Increase the number of washes or add low concentrations of detergents (0.1-0.3% Triton X-100) to reduce background.

• Titrate antibody concentration: Test a range of antibody dilutions to find the optimal concentration that maximizes specific signal while minimizing background.

• Pre-adsorption: Pre-incubate the antibody with unmodified histone peptides to neutralize antibodies that may cross-react with unmodified histones.

• Consider fixation methods: For immunofluorescence applications, optimize fixation protocols. Successful immunofluorescence has been performed with 4% formaldehyde fixation for 10 minutes followed by blocking with PBS/TX-100 containing 5% normal goat serum and 1% BSA .

• Validate with knockout/knockdown controls: When available, use samples from cells where the histone acetyltransferase responsible for K120 acetylation has been depleted as negative controls.

These strategies can significantly improve experimental outcomes and data reliability when working with histone modification antibodies.

How can ChIP-seq data for HIST1H2BC acetylation be appropriately analyzed to identify regulatory elements?

Analysis of ChIP-seq data for Acetyl-HIST1H2BC (K120) to identify regulatory elements requires a structured computational approach:

• Quality control and alignment: After sequencing, perform quality filtering of reads before aligning to the reference genome using algorithms like BWA, as used in similar histone modification studies .

• Peak calling: Use specialized algorithms (MACS2, SICER) optimized for histone modifications, which typically produce broader peaks than transcription factor binding sites.

• Genomic distribution analysis: Analyze the distribution of acetylation signals across genomic features (promoters, enhancers, gene bodies) to understand the modification's preferential localization.

• Integration with gene expression data: Correlate acetylation patterns with RNA-seq data to identify relationships between K120 acetylation and transcriptional activity.

• Motif analysis: Identify DNA sequence motifs enriched in regions with high acetylation to identify potential transcription factor binding sites.

• Comparative analysis: Compare acetylation patterns across different cell types or experimental conditions to identify cell-type-specific regulatory elements.

• Visualization: Generate genome browser tracks to visualize enrichment along chromosomes and at specific loci, similar to the visualization of enrichment along chromosome regions shown in ChIP-seq analyses of similar histone modifications .

Analysis of enrichment profiles around the transcription start sites (TSS) of genes is particularly informative for identifying promoter-associated regulatory elements, while intergenic peaks may represent enhancers or other regulatory regions.

How does HIST1H2BC K120 acetylation compare functionally with K12 acetylation in gene regulation?

The functional distinction between H2B acetylation at K120 versus K12 represents an important aspect of the histone code:

• Genomic localization: K120 acetylation is often enriched in gene bodies of actively transcribed genes and correlates with transcriptional elongation, while K12 acetylation is typically associated with promoter regions and transcription initiation.

• Enzyme specificity: Different histone acetyltransferases (HATs) target these sites: K120 is primarily acetylated by p300/CBP, while K12 may be targeted by other HATs including GCN5/PCAF family members.

• Functional outcomes: K120 acetylation has been implicated in facilitating nucleosome reassembly following RNA polymerase II passage, whereas K12 acetylation appears more involved in nucleosome destabilization at promoters to facilitate transcription initiation.

• Temporal dynamics: Studies suggest K12 acetylation often precedes K120 acetylation during gene activation, reflecting the progression from transcription initiation to elongation.

• Interplay with other modifications: K120 acetylation can influence H2B ubiquitination at K120/123, creating a dynamic interplay between different modifications at the same residue, while K12 acetylation may cooperate with other promoter-enriched marks.

Understanding these distinct roles requires experimental approaches that specifically distinguish between these modifications, emphasizing the importance of highly specific antibodies like those described in the search results .

What is the relationship between histone H2B acetylation and other epigenetic modifications in regulating chromatin accessibility?

Histone H2B acetylation functions within a complex network of epigenetic modifications that collectively regulate chromatin accessibility:

• Crosstalk with methylation: H2B acetylation can influence H3K4 and H3K79 methylation through a trans-histone pathway, where H2B modifications affect modifications on H3 within the same nucleosome. This relationship is critical for proper transcriptional regulation.

• Coordination with chromatin remodeling: Acetylated H2B can recruit ATP-dependent chromatin remodeling complexes that physically reposition nucleosomes to increase DNA accessibility.

• Relationship with DNA methylation: Regions with high H2B acetylation typically show low DNA methylation levels, demonstrating the reciprocal relationship between these modifications in maintaining active chromatin states.

• Pioneer factor binding: Acetylation of H2B can facilitate binding of pioneer transcription factors that can access partially closed chromatin and promote further opening.

• Nucleosome stability: H2B acetylation, particularly at multiple sites, can destabilize nucleosome structure by neutralizing positive charges that interact with the negatively charged DNA backbone.

• Histone variant incorporation: Acetylation patterns on H2B can influence the incorporation of histone variants, further altering chromatin properties and accessibility.

How do the patterns of HIST1H2BC acetylation change during cellular differentiation and development?

The dynamics of HIST1H2BC acetylation during cellular differentiation and development follow specific patterns that reflect changing gene expression programs:

• Developmental reprogramming: During embryonic development, global reorganization of histone acetylation patterns occurs, with H2B acetylation increasing at developmentally regulated genes as they become activated in specific lineages.

• Cell-type specific profiles: Differentiated cell types exhibit distinct H2B acetylation signatures that correlate with their gene expression profiles and cellular functions. This can be observed through ChIP-seq analysis comparing different cell types or developmental stages.

• Temporal regulation: Time-course analyses during differentiation processes reveal that H2B acetylation changes can both precede and follow transcriptional changes, suggesting both instructive and reinforcing roles.

• Enhancer priming: During differentiation, H2B acetylation often appears at enhancers before gene activation, preparing chromatin for subsequent transcription factor binding.

• Relationship to bivalent domains: In stem cells, some developmental genes contain bivalent domains with both activating and repressive marks; during differentiation, H2B acetylation increases at genes resolving toward activation.

• Epigenetic memory: Patterns of H2B acetylation can be maintained through cell divisions, contributing to epigenetic memory that preserves cell identity.

These dynamic changes can be studied using ChIP-seq with antibodies specific to acetylated H2B at different time points during differentiation, providing insights into the epigenetic regulation of development .

What are the advantages and limitations of using polyclonal versus monoclonal antibodies for detecting histone acetylation?

Understanding the tradeoffs between polyclonal and monoclonal antibodies is crucial for experimental design:

How can mass spectrometry be used to validate and complement antibody-based detection of histone acetylation?

Mass spectrometry (MS) provides powerful complementary approaches to antibody-based detection of histone acetylation:

• Antibody validation: MS can verify antibody specificity by analyzing immunoprecipitated samples to confirm the presence of the target modification and quantify potential cross-reactivity with other modifications.

• Combinatorial modifications: Unlike antibodies that typically detect single modifications, MS can identify multiple modifications on the same histone molecule, revealing combinatorial patterns that may have functional significance.

• Quantitative analysis: MS enables precise quantification of modification stoichiometry (percentage of histones modified at a specific site), providing information not readily obtainable with antibodies.

• Novel modification discovery: MS can identify previously unknown modifications that may then become targets for antibody development.

• Workflow integration: An integrated workflow might involve:

  • Initial ChIP-seq with Acetyl-HIST1H2BC (K120) antibody to identify genomic locations

  • MS analysis of histones from the same samples to quantify modification levels

  • Targeted MS on immunoprecipitated chromatin to validate antibody specificity

• Technical considerations: For MS analysis of histones, specialized protocols for histone extraction, chemical derivatization (to improve peptide properties for MS), and targeted acquisition methods are typically employed.

This combined approach provides more comprehensive insights into histone modifications than either technique alone, enhancing the reliability and depth of epigenetic research findings.

What are the considerations for using HIST1H2BC acetylation antibodies in single-cell epigenomic techniques?

Adapting histone acetylation antibodies for single-cell applications requires special considerations:

• Sensitivity optimization: Single-cell techniques require exceptionally high antibody sensitivity due to limited material. Consider using signal amplification methods such as tyramide signal amplification for immunofluorescence or highly sensitive detection systems for ChIP applications.

• Antibody concentration: Typically higher antibody concentrations are needed for single-cell applications compared to bulk assays. Careful titration experiments should be performed to determine optimal concentrations.

• Background reduction: Minimizing non-specific binding is crucial in single-cell applications where signal-to-noise ratio is critical. Consider additional blocking steps and more stringent washing protocols.

• Combinatorial approaches: For studying HIST1H2BC acetylation in single cells, consider techniques like:

  • CUT&Tag or CUT&RUN adapted for single cells

  • Single-cell ChIP-seq with antibody barcoding

  • Imaging-based approaches like single-cell immunofluorescence combined with microscopy

• Validation strategies: Validate findings from single-cell techniques using orthogonal approaches such as:

  • Comparing pooled single-cell data with bulk assays

  • Using cell populations with known acetylation patterns as controls

  • Confirming key findings with alternative antibodies or methods

• Data analysis considerations: Single-cell epigenomic data requires specialized analytical approaches to account for technical noise, sparse data matrices, and cellular heterogeneity.

These adaptations enable researchers to study the heterogeneity of histone acetylation patterns within cell populations, providing insights into epigenetic variation that would be masked in bulk analyses.

How can Acetyl-HIST1H2BC antibodies be used to investigate epigenetic dysregulation in cancer?

Acetyl-HIST1H2BC antibodies provide valuable tools for investigating cancer epigenetics:

• Cancer-specific acetylation patterns: ChIP-seq using these antibodies can identify altered H2B acetylation patterns in cancer cells compared to normal cells, potentially revealing cancer-specific epigenetic signatures.

• Biomarker development: Quantification of H2B acetylation levels in patient samples using these antibodies may help identify prognostic or diagnostic biomarkers. Immunohistochemistry protocols can be adapted from the ICC/IF methods described in the search results .

• Drug response studies: Acetyl-HIST1H2BC antibodies can monitor changes in acetylation patterns following treatment with epigenetic drugs, such as histone deacetylase inhibitors (e.g., sodium butyrate mentioned in result ) or histone acetyltransferase inhibitors.

• Mechanistic investigations: These antibodies enable studies on how oncogenes or tumor suppressors influence the epigenetic landscape through changes in histone acetylation.

• Therapeutic target identification: Regions showing aberrant H2B acetylation in cancer cells can be explored as potential therapeutic targets, with the antibodies being used to validate the specificity of drugs targeting these modifications.

• Cancer heterogeneity: When adapted to single-cell techniques, these antibodies can reveal epigenetic heterogeneity within tumors, potentially identifying treatment-resistant subpopulations.

The experimental approaches would be similar to those described earlier but applied specifically to cancer cell lines, patient-derived xenografts, or clinical samples to understand the role of histone acetylation in cancer development and progression.

What are the best practices for using histone modification antibodies in fixed clinical samples?

Applying histone modification antibodies to clinical samples presents unique challenges requiring specific methodological considerations:

• Fixation optimization: Formalin fixation can mask epitopes and hinder antibody binding. For FFPE (formalin-fixed paraffin-embedded) samples, antigen retrieval methods should be optimized specifically for histone modifications:

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0) or Tris-EDTA (pH 9.0)

  • Enzymatic retrieval methods may be less effective for nuclear proteins

  • Retrieval times may need to be extended (20-40 minutes)

• Section thickness and preparation: 4-5 μm sections are typically optimal for immunohistochemistry with histone antibodies.

• Blocking and permeabilization: Enhanced blocking protocols (e.g., using both serum and BSA as in the protocols mentioned in ) and adequate permeabilization are crucial for reducing background in tissue samples.

• Antibody validation in fixed tissues: Perform validation specifically in fixed tissues, as antibodies that work well in cell lines may perform differently in FFPE samples.

• Controls:

  • Include tissue samples known to have high and low levels of the target modification

  • Use peptide competition assays to confirm specificity in tissue context

  • Consider tissue from patients treated with HDAC inhibitors as positive controls

• Signal detection and quantification:

  • Employ standardized scoring systems for immunohistochemistry

  • Consider automated image analysis for more objective quantification

  • Use multiplex immunofluorescence to correlate histone modifications with other markers

• Protocol standardization: Maintain consistent protocols across clinical samples to enable reliable comparisons, particularly in biomarker studies.

These practices help ensure reliable detection of histone modifications in clinical samples, facilitating translation of epigenetic research to clinical applications.