Acetyl-HIST1H2BB (K16) Antibody

What is the Acetyl-HIST1H2BB (K16) Antibody and what specific target does it recognize?

The Acetyl-HIST1H2BB (K16) Antibody is a polyclonal antibody raised in rabbits that specifically recognizes the acetylated lysine 16 residue of Histone H2B type 1-B protein in humans. This antibody binds to the post-translational modification site on HIST1H2BB, also known by several synonyms including H2BFF Histone H2B type 1-B, Histone H2B.1, Histone H2B.f, and H2B/f . The antibody was developed using a synthetic peptide sequence surrounding the acetylated lysine 16 position derived from human Histone H2B type 1-B as the immunogen . This specific modification is part of the histone code that regulates chromatin structure and accessibility, playing crucial roles in transcriptional regulation, DNA repair, replication, and chromosomal stability .

What are the validated applications for this antibody in research settings?

The Acetyl-HIST1H2BB (K16) Antibody has been validated for multiple research applications:

This versatility allows researchers to employ the antibody across multiple experimental platforms, enabling comprehensive investigation of histone acetylation patterns and their functional consequences in various cellular contexts .

What is the scientific significance of H2B K16 acetylation in epigenetic research?

Histone H2B lysine 16 acetylation represents a critical epigenetic modification that influences chromatin structure and gene regulation. As a core component of nucleosomes, H2B and its modifications directly impact how DNA is packaged and accessed by transcriptional machinery . This specific acetylation mark contributes to the histone code that regulates chromatin accessibility, enabling or restricting cellular processes that require DNA as a template . Scientifically, studying this modification provides insights into how epigenetic changes influence gene expression patterns, cellular differentiation, and disease progression. The Acetyl-HIST1H2BB (K16) Antibody thus serves as an essential tool for researchers investigating fundamental mechanisms of epigenetic regulation and their biological consequences in human cells .

What are the recommended sample preparation techniques to maximize antibody performance?

For optimal performance of the Acetyl-HIST1H2BB (K16) Antibody across different applications, sample preparation should be carefully optimized:

For Western Blotting:

• Extract histones using acid extraction (0.2N HCl) to efficiently recover histones

• Use freshly prepared samples when possible, or store protein extracts with protease and deacetylase inhibitors

• Include sodium butyrate (5-10 mM) in lysis buffers to prevent deacetylation during sample preparation

• Denature samples completely in loading buffer containing SDS and reducing agents

• Transfer to PVDF membranes is typically more effective than nitrocellulose for histone proteins

For Immunofluorescence/Immunocytochemistry:

• Fix cells with 4% paraformaldehyde for 10-15 minutes at room temperature

• Permeabilize with 0.1-0.5% Triton X-100 for nuclear proteins

• Block with 5% normal serum (from the species of secondary antibody) to reduce background

• Include acetylation-preserving agents in fixation buffers

• Consider epitope retrieval methods if signal is weak

For ChIP applications:

• Cross-link chromatin with 1% formaldehyde for 10 minutes at room temperature

• Ensure chromatin is sheared to appropriate fragment sizes (200-500 bp)

• Pre-clear chromatin with protein A/G beads before antibody addition

• Include input controls and negative controls (IgG)

Proper sample preparation significantly impacts experimental success, particularly for detecting post-translational modifications like acetylation that can be dynamically regulated in cells .

What controls should be included when designing experiments with this antibody?

Proper experimental controls are essential for validating results obtained with the Acetyl-HIST1H2BB (K16) Antibody:

Essential Controls:

• Positive Control:

  • Known cell lines or tissues with high levels of H2B K16 acetylation

  • Cells treated with histone deacetylase inhibitors (e.g., TSA, SAHA)

  • Recombinant acetylated H2B peptide (if available)

• Negative Controls:

  • Isotype control (rabbit IgG) at equivalent concentration

  • Competitive peptide blocking (using the immunizing peptide)

  • Samples treated with histone acetyltransferase inhibitors

  • CRISPR-modified cells lacking the target modification

• Antibody Validation Controls:

  • Peptide competition assay to confirm specificity

  • Secondary antibody-only control to assess background

  • Acetylation site mutants (K16R) if available in your experimental system

• Technical Controls:

  • Loading controls for Western blots (total H2B or other stable proteins)

  • Nuclear counterstains for IF/ICC (DAPI, Hoechst)

  • Input chromatin samples for ChIP experiments (1-10% of starting material)

Including these controls allows proper interpretation of results and validation of antibody specificity, particularly important when studying post-translational modifications that may be present at low abundance or in specific cellular contexts .

How should the antibody be stored and handled to maintain optimal activity?

Proper storage and handling of the Acetyl-HIST1H2BB (K16) Antibody are critical for maintaining its specificity and sensitivity:

Storage Recommendations:

• Store undiluted antibody at -20°C or -80°C for long-term storage

• Avoid repeated freeze-thaw cycles that can degrade antibody quality and performance

• Consider aliquoting the stock antibody solution into single-use volumes upon receipt

• For short-term storage (up to one week), the antibody can be kept at 4°C

Handling Guidelines:

• The antibody is typically supplied in a buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative

• Allow the antibody to equilibrate to room temperature before opening the vial

• Centrifuge briefly before use to collect liquid at the bottom of the tube

• Use clean, DNase/RNase-free pipette tips when handling

• Return to recommended storage temperature promptly after use

• For diluted working solutions, prepare fresh when possible or store with carrier protein (BSA)

Shipping and Transportation:

• The antibody is typically shipped with cooling packs or on dry ice

• Upon receipt, verify the integrity of the package and immediately transfer to appropriate storage

Adherence to these storage and handling recommendations helps ensure consistent antibody performance across experiments and extends the usable lifetime of this valuable research reagent .

How can Acetyl-HIST1H2BB (K16) Antibody be integrated into multi-omics approaches for comprehensive epigenetic profiling?

The Acetyl-HIST1H2BB (K16) Antibody can serve as a powerful component in multi-omics research strategies to create comprehensive epigenetic profiles:

ChIP-seq Integration:

• The antibody can be used in ChIP-seq experiments to map genome-wide distribution of H2B K16 acetylation

• Combined with RNA-seq from the same samples, researchers can correlate H2B K16ac patterns with transcriptional activity

• Sequential ChIP (re-ChIP) can be performed to identify genomic regions containing multiple histone modifications

• Integration with ATAC-seq or DNase-seq data reveals relationships between this acetylation mark and chromatin accessibility

Mass Spectrometry Approaches:

• Use the antibody for immunoprecipitation followed by mass spectrometry to identify proteins that interact with acetylated H2B

• Quantitative proteomics can reveal changes in acetylation levels under different experimental conditions

• This approach enables identification of writer/reader/eraser enzymes specific to this modification

Single-Cell Applications:

• Adapt ChIP protocols using this antibody for CUT&Tag or CUT&RUN methods for increased sensitivity

• Combine with single-cell RNA-seq data for correlation between H2B K16ac and gene expression at single-cell resolution

• Use in imaging mass cytometry for spatial context of histone modifications

Temporal and Spatial Studies:

• Time-course experiments with the antibody can track dynamic changes in H2B K16 acetylation

• Combined with chromosome conformation capture techniques (Hi-C, 4C) to correlate histone acetylation with 3D genome organization

This integrated approach provides a more complete understanding of how H2B K16 acetylation contributes to the complex regulatory network controlling gene expression and chromatin structure in various biological contexts .

What are the methodological considerations for studying the relationship between Acetyl-HIST1H2BB (K16) and gene expression regulation?

When investigating the relationship between H2B K16 acetylation and gene expression regulation, researchers should consider several methodological approaches and technical considerations:

Sequential ChIP-seq and RNA-seq Analysis:

• Perform ChIP-seq with the Acetyl-HIST1H2BB (K16) Antibody to identify genomic regions enriched for this modification

• Follow with RNA-seq on the same biological samples to correlate acetylation patterns with transcriptional activity

• Use bioinformatic tools to identify motifs in regions with high H2B K16ac enrichment to identify potential regulatory factors

• Consider time-course experiments to track dynamic changes in both acetylation and transcription

Genomic Context Analysis:

• Determine whether H2B K16ac is enriched at specific genomic features (promoters, enhancers, gene bodies)

• Analyze the co-occurrence with other histone modifications using additional ChIP experiments

• Investigate the relationship with chromatin accessibility through ATAC-seq or DNase-seq

• Examine correlation with CpG island distribution and DNA methylation patterns

Functional Validation Experiments:

• Use CRISPR-Cas9 to target histone acetyltransferases or deacetylases specific to H2B K16

• Employ site-specific histone mutants (K16R to prevent acetylation or K16Q to mimic acetylation)

• Utilize specific inhibitors of enzymes regulating this modification

• Perform reporter assays with constructs containing regions identified in ChIP-seq experiments

Technical Considerations:

• Include appropriate controls for ChIP-seq (input DNA, IgG controls)

• Validate ChIP-seq peaks with ChIP-qPCR for selected regions

• Normalize RNA-seq data appropriately to account for technical and biological variation

• Consider cell-type specific effects and heterogeneity in cell populations

These methodological considerations ensure robust investigation of the functional relationship between H2B K16 acetylation and transcriptional regulation, providing insights into the mechanistic role of this modification in gene expression control .

How can this antibody be used to study the dynamics of histone acetylation during cellular processes and disease states?

The Acetyl-HIST1H2BB (K16) Antibody provides a valuable tool for investigating temporal changes in histone acetylation during various cellular processes and disease states:

Cell Cycle and Differentiation Studies:

• Synchronize cells at different cell cycle phases and use the antibody in Western blots or immunofluorescence to track H2B K16ac changes

• In differentiation models, perform time-course ChIP-seq to map genome-wide redistribution of this acetylation mark

• Combine with EdU labeling or PCNA staining to correlate acetylation with DNA replication timing

• Use flow cytometry with this antibody to quantify acetylation levels in subpopulations of cells

Disease Model Applications:

• Compare H2B K16ac patterns between normal and disease tissues using immunohistochemistry

• Perform ChIP-seq in patient-derived samples to identify disease-specific alterations in acetylation patterns

• Use in drug screening platforms to identify compounds that modulate this specific acetylation mark

• Investigate changes in response to environmental stressors or signaling pathway activation

Live-Cell Dynamics:

• Develop proximity ligation assays using this antibody to visualize acetylation in fixed cells with higher sensitivity

• Adapt for FRAP (Fluorescence Recovery After Photobleaching) studies to examine turnover rates of acetylated histones

• Combine with optogenetic tools to induce targeted histone modifications and monitor consequences

Methodological Protocol Example for Temporal Studies:

• Treat cells with stimulus of interest at multiple time points (0, 15, 30, 60 min, etc.)

• Process parallel samples for:

  • Western blot with the Acetyl-HIST1H2BB (K16) Antibody (1:500 dilution)

  • ChIP-qPCR at candidate regulatory regions (use 5μg antibody per reaction)

  • Immunofluorescence to visualize nuclear distribution changes

• Quantify changes relative to time zero and normalize to appropriate controls

• For selected time points, perform ChIP-seq to obtain genome-wide profiles

This approach enables comprehensive characterization of H2B K16 acetylation dynamics in response to various stimuli, providing insights into the temporal regulation of this epigenetic modification in normal and pathological conditions .

What are common issues encountered when using Acetyl-HIST1H2BB (K16) Antibody and how can they be resolved?

Researchers may encounter several technical challenges when working with the Acetyl-HIST1H2BB (K16) Antibody. Here are common issues and their solutions:

Weak or No Signal:

• Possible Causes: Insufficient antibody concentration, epitope masking, low abundance of modification, degradation of acetyl mark

• Solutions:

  • Increase antibody concentration within recommended range (1:50-1:200 for IF/ICC)

  • Include deacetylase inhibitors (e.g., sodium butyrate, TSA) in sample preparation

  • Try different epitope retrieval methods for fixed tissues

  • Use fresh samples and minimize processing time

  • Validate antibody activity with positive control samples

High Background:

• Possible Causes: Insufficient blocking, too high antibody concentration, cross-reactivity

• Solutions:

  • Optimize blocking conditions (increase blocking time/concentration)

  • Decrease primary antibody concentration

  • Include additional wash steps with increased stringency

  • Pre-absorb antibody with acetylated peptide library excluding the target

  • Use more specific secondary antibodies

Inconsistent ChIP Results:

• Possible Causes: Inefficient chromatin shearing, sub-optimal antibody amount, variable crosslinking

• Solutions:

  • Optimize sonication conditions for consistent fragment sizes

  • Titrate antibody amount (typically 3-5μg per ChIP reaction)

  • Standardize crosslinking time and conditions

  • Include spike-in controls for normalization across experiments

  • Use sequential ChIP approaches for increased specificity

Cross-Reactivity Issues:

• Possible Causes: Antibody recognizing similar acetylation sites on other histones

• Solutions:

  • Perform peptide competition assays with related acetylated peptides

  • Include appropriate negative controls (non-acetylated samples)

  • Validate with orthogonal techniques (mass spectrometry)

  • Consider using monoclonal antibodies for highest specificity if available

Careful optimization of experimental conditions and inclusion of appropriate controls help overcome these technical challenges and ensure reliable results when working with this histone modification-specific antibody .

How does sample preparation affect the detection of H2B K16 acetylation in different experimental contexts?

Sample preparation significantly impacts the detection of H2B K16 acetylation across different experimental platforms. Understanding these effects is critical for obtaining reliable and reproducible results:

Protein Extraction for Western Blotting:

• Critical Factors:

  • Histone acetylation is dynamically regulated and susceptible to rapid deacetylation during extraction

  • Acidic extraction methods (0.2N HCl or 0.4N H2SO4) efficiently recover histones but may affect some modifications

  • Inclusion of deacetylase inhibitors is essential (sodium butyrate, TSA, nicotinamide)

  • Sample handling time should be minimized with cold temperature processing

  • Protease inhibitors must be included to prevent degradation

Fixation Effects on Immunofluorescence/Immunocytochemistry:

• Comparative Analysis:

• Over-fixation can mask epitopes and reduce signal intensity

• Permeabilization conditions must be optimized for nuclear proteins (0.2% Triton X-100 for 10 minutes typically suitable)

Chromatin Preparation for ChIP:

• Cross-linking time affects epitope accessibility (standard: 1% formaldehyde for 10 minutes)

• Over-crosslinking can reduce antibody binding efficiency

• Sonication conditions impact fragment size and epitope integrity

• Native ChIP (without crosslinking) may preserve some modifications better but loses transient interactions

• Use of micrococcal nuclease in combination with sonication can improve chromatin fragmentation consistency

Tissue Processing Effects:

• Paraffin embedding can reduce acetylation detection (requires optimized antigen retrieval)

• Frozen sections better preserve modifications but have poorer morphology

• Post-mortem interval significantly affects acetylation stability

• FFPE tissues may require specialized recovery techniques for modifications

Understanding these sample preparation variables enables researchers to select appropriate processing methods for their specific experimental goals and optimize detection of H2B K16 acetylation across different applications .

What factors influence the specificity and sensitivity of the Acetyl-HIST1H2BB (K16) Antibody in different applications?

Multiple factors influence the specificity and sensitivity of the Acetyl-HIST1H2BB (K16) Antibody across different experimental applications:

Antibody Characteristics:

• Polyclonal Nature: As a polyclonal antibody raised in rabbits, batch-to-batch variation may occur; validation with each new lot is recommended

• Epitope Recognition: The antibody targets a specific peptide sequence surrounding acetylated K16, but similar sequences in other histones may cause cross-reactivity

• Affinity Purification: The antibody undergoes antigen affinity purification to enhance specificity, but optimization for each application remains necessary

Technical Factors Affecting Performance:

Biological Variables:

• Abundance of Modification: H2B K16 acetylation levels vary across cell types and conditions, affecting detection sensitivity

• Competing Modifications: Adjacent or nearby histone modifications may interfere with antibody binding

• Protein Complexes: Interaction partners may mask the epitope in certain experimental conditions

• Dynamic Regulation: The rapid turnover of acetylation marks necessitates careful timing of experiments

Enhancing Specificity Strategies:

• Peptide competition assays to confirm specificity for the acetylated vs. non-acetylated form

• Use of HDAC inhibitors to increase acetylation signal for positive controls

• Comparison with other commercially available antibodies targeting the same modification

• Validation in systems with genetic manipulation of acetyltransferases/deacetylases

• Secondary confirmation with mass spectrometry when possible

Researchers should consider these factors when designing experiments and interpreting results, particularly when studying subtle changes in histone acetylation patterns or when comparing results across different experimental platforms .

How is the Acetyl-HIST1H2BB (K16) Antibody contributing to our understanding of epigenetic mechanisms in disease?

The Acetyl-HIST1H2BB (K16) Antibody has become an important tool in uncovering the role of histone H2B K16 acetylation in various disease contexts:

Cancer Epigenetics:

• Researchers have utilized this antibody to demonstrate altered H2B K16 acetylation patterns in multiple cancer types

• Studies have revealed correlations between changes in this modification and tumor progression or treatment response

• The antibody has helped identify potential epigenetic biomarkers for cancer diagnosis and prognosis

• Investigation of histone acetyltransferases and deacetylases that regulate this site has uncovered potential therapeutic targets

Neurodegenerative Disorders:

• Studies using this antibody have shown dysregulation of H2B K16 acetylation in models of Alzheimer's and Parkinson's diseases

• Researchers have connected changes in this modification with altered neuronal gene expression profiles

• The antibody has facilitated investigation of epigenetic changes during disease progression

Inflammatory and Autoimmune Conditions:

• The antibody has helped reveal how H2B K16 acetylation regulates inflammatory gene expression programs

• Studies have shown dynamic changes in this modification during immune cell activation and differentiation

• Research has connected alterations in H2B acetylation with autoimmune disease susceptibility

Developmental Disorders:

• Investigations using this antibody have uncovered the role of H2B K16 acetylation in normal development and developmental disorders

• The antibody has helped characterize epigenetic reprogramming events during cellular differentiation

Emerging Research Areas:

• Integration of H2B K16 acetylation data with other epigenetic modifications to build comprehensive disease models

• Investigation of this modification in response to environmental exposures and stressors

• Studies examining the transgenerational inheritance of epigenetic patterns involving this modification

Through these applications, the Acetyl-HIST1H2BB (K16) Antibody continues to advance our understanding of epigenetic dysregulation in human diseases and identify potential targets for epigenetic therapies .

What emerging technologies could enhance the application of this antibody in future epigenetic research?

Emerging technologies are expanding the potential applications of the Acetyl-HIST1H2BB (K16) Antibody in epigenetic research:

Advanced Imaging Technologies:

• Super-resolution Microscopy: Techniques like STORM, PALM, and STED enable visualization of H2B K16 acetylation distribution at nanometer resolution, revealing subnuclear localization patterns previously undetectable

• Live-cell Imaging Adaptations: Development of acetylation-specific intrabodies or nanobodies derived from this antibody sequence could enable real-time tracking of acetylation dynamics

• Spatial Omics Integration: Combining immunofluorescence using this antibody with spatial transcriptomics to correlate acetylation patterns with gene expression in preserved tissue architecture

Single-Cell Epigenomic Methods:

• Single-Cell ChIP Technologies: Adaptations of this antibody for ultra-low input ChIP protocols enable acetylation profiling in rare cell populations

• CUT&Tag/CUT&RUN Protocols: These antibody-directed transposase-based methods offer increased sensitivity and reduced background for H2B K16ac profiling

• Single-Cell Multi-omics: Integration with single-cell RNA-seq and ATAC-seq data for comprehensive epigenetic-transcriptomic correlation at single-cell resolution

Massively Parallel Reporter Assays:

• High-throughput functional testing of genomic regions identified by ChIP-seq with this antibody

• Systematic analysis of how H2B K16 acetylation affects enhancer activity across thousands of regulatory elements

Computational and AI-Based Approaches:

• Machine learning algorithms to predict H2B K16 acetylation patterns from DNA sequence features

• Network analysis integrating ChIP-seq data from this antibody with other epigenetic marks to infer regulatory relationships

• Predictive modeling of acetylation dynamics in response to cellular perturbations

Engineered Epigenetic Modifiers:

• Adapting the antibody's binding domain for targeted epigenetic editing technologies (dCas9-based systems)

• Development of synthetic readers or erasers specific to this modification for functional studies

• Optogenetic control of site-specific histone acetylation to study temporal dynamics

These emerging technologies promise to dramatically expand our understanding of H2B K16 acetylation's role in chromatin biology and gene regulation, with the Acetyl-HIST1H2BB (K16) Antibody serving as a fundamental tool in these advanced applications .

What are the most significant recent findings regarding H2B K16 acetylation in chromatin regulation and nuclear architecture?

Recent research utilizing the Acetyl-HIST1H2BB (K16) Antibody has revealed several significant insights about the role of H2B K16 acetylation in chromatin organization and nuclear function:

Chromatin Accessibility and Nucleosome Stability:

• Studies have demonstrated that H2B K16 acetylation alters nucleosome stability and promotes a more accessible chromatin state

• This modification influences nucleosome sliding rates and affects higher-order chromatin folding

• Research shows that H2B K16ac can function cooperatively with other histone modifications to establish permissive chromatin domains

• The antibody has helped map the genome-wide distribution of this modification relative to open chromatin regions identified by ATAC-seq

Transcriptional Regulation Mechanisms:

• Recent findings reveal that H2B K16 acetylation is preferentially enriched at the promoters and enhancers of actively transcribed genes

• This modification has been shown to facilitate the recruitment of specific transcription factors and co-activators

• Studies using the antibody have identified dynamic changes in H2B K16ac during transcriptional responses to various stimuli

• Research has uncovered crosstalk between H2B K16 acetylation and other histone modifications in coordinating gene expression programs

Nuclear Compartmentalization:

• Advanced imaging with this antibody has revealed non-random distribution of H2B K16ac within the nucleus

• This modification shows distinct patterns of enrichment or depletion in specific nuclear compartments (e.g., nuclear speckles, nucleolus periphery)

• Studies have connected H2B K16 acetylation patterns with chromosomal territories and TAD (Topologically Associated Domain) boundaries

• Research suggests this modification may contribute to phase separation properties of chromatin domains

Cell Cycle Dynamics:

• Investigations have revealed cell cycle-specific fluctuations in H2B K16 acetylation levels

• This modification shows redistribution patterns during mitosis and early G1

• Studies suggest roles in replication timing and post-replication chromatin assembly

• The antibody has been instrumental in tracking inheritance patterns of this modification through cell divisions

Writers, Readers and Erasers:

• Recent work has identified specific histone acetyltransferases (HATs) and histone deacetylases (HDACs) that regulate H2B K16 acetylation

• Novel protein complexes that specifically recognize this modification have been characterized

• Research has revealed context-dependent recruitment of these enzymes to regulate H2B K16ac levels in response to cellular signaling

These findings highlight the diverse and critical roles of H2B K16 acetylation in chromatin biology and nuclear function, with the Acetyl-HIST1H2BB (K16) Antibody serving as an essential tool in advancing our understanding of these complex epigenetic mechanisms .