Acetyl-HIST1H2BC (K15) Antibody

What is the Acetyl-HIST1H2BC (K15) Antibody and what does it detect?

The Acetyl-HIST1H2BC (K15) Antibody (such as PACO60597) is a polyclonal antibody raised in rabbits that specifically recognizes and binds to the acetylated form of histone variant Hist1H2BC at lysine 15 position. This antibody detects a post-translational modification (PTM) that plays a critical role in epigenetic regulation. The acetylation of Hist1H2BC at K15 is known to be associated with transcriptional activation by influencing chromatin structure, making it accessible to transcription machinery . The antibody specifically targets the peptide sequence surrounding the acetylated Lys-15 site derived from Human Histone H2B type 1-C/E/F/G/I .

What are the validated applications for Acetyl-HIST1H2BC (K15) Antibody?

The Acetyl-HIST1H2BC (K15) Antibody has been validated for multiple research applications with specific recommended dilutions:

These applications allow researchers to study the presence, abundance, and localization of acetylated Hist1H2BC (K15) in various experimental contexts . When designing experiments, it is important to validate these dilutions in your specific experimental setup before proceeding with full-scale studies.

How should Acetyl-HIST1H2BC (K15) Antibody be stored and handled?

For optimal performance and longevity, the Acetyl-HIST1H2BC (K15) Antibody should be stored according to the following guidelines:

The antibody is provided in liquid form with a storage buffer containing 0.03% Proclin 300 as a preservative, 50% Glycerol, and 0.01M PBS at pH 7.4 . Upon receipt, the antibody should be stored at -20°C or -80°C . Repeated freeze-thaw cycles should be avoided as they can degrade antibody quality and affect binding specificity . For working solutions, small aliquots should be prepared and stored separately to minimize freeze-thaw cycles. Before use, the antibody should be allowed to reach room temperature and gently mixed (not vortexed) to ensure homogeneity.

What is the biological significance of H2B acetylation at lysine 15?

The acetylation of Hist1H2BC at lysine 15 represents a critical epigenetic modification with profound biological implications. This modification is part of the "histone code" that regulates DNA accessibility to cellular machinery . Specifically, H2B K15 acetylation:

• Contributes to chromatin decondensation by neutralizing the positive charge of lysine, weakening histone-DNA interactions

• Creates binding sites for bromodomain-containing proteins that facilitate transcription factor recruitment

• Plays a role in transcriptional activation of specific genes

• May be involved in DNA repair processes and replication

The presence of H2B K15 acetylation is generally associated with active gene transcription and open chromatin states . Understanding the dynamics of this modification provides insights into gene regulation mechanisms and may have implications for therapeutic development targeting epigenetic modifications in diseases such as cancer and neurological disorders .

How can cross-reactivity with other histone acetylation marks be assessed and mitigated?

Cross-reactivity represents a significant challenge when working with histone modification antibodies due to the high sequence similarity between different histone variants and modification sites. For the Acetyl-HIST1H2BC (K15) Antibody, cross-reactivity assessment and mitigation should include:

Peptide competition assays can be employed where the antibody is pre-incubated with excess acetylated and non-acetylated peptides corresponding to the K15 site and similar sites on other histones. A true specific antibody will show reduced signal when pre-incubated with the acetylated K15 peptide but not with other peptides .

Western blotting with recombinant histones containing defined modifications can help determine specificity. Research has shown that some H2B acetylation antibodies may cross-react with similar epitopes, particularly between K5 and K27 due to sequence similarities . For example, studies have found that certain H2BK5ac antibodies cross-reacted with acetylated H3 peptides .

ChIP-seq correlation analysis can reveal potential cross-reactivity issues. As noted in recent studies, antibodies that demonstrate unusually high correlation with modifications at different sites may be cross-reacting . Implementing a dual ChIP approach (sequential immunoprecipitation with two different antibodies) can improve specificity when cross-reactivity is a concern.

What are the optimal ChIP-seq protocols for detecting Acetyl-HIST1H2BC (K15) in various cell types?

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a powerful technique for genome-wide profiling of histone modifications. For optimal detection of Acetyl-HIST1H2BC (K15), consider the following protocol optimizations:

Cell-type specific considerations:
Different cell types may require adjusted fixation conditions. For adherent cells like 293 cells (commonly used with this antibody ), standard 1% formaldehyde for 10 minutes at room temperature is typically effective. For suspension cells or tissues, optimization of fixation time may be necessary.

Chromatin preparation:
Sonication conditions should be optimized to generate fragments of 200-500bp. Over-sonication can destroy epitopes, while under-sonication reduces resolution. The addition of SDS to a final concentration of 1% in lysis buffers helps expose epitopes in compact chromatin.

Immunoprecipitation:
For the Acetyl-HIST1H2BC (K15) Antibody, use 2-5μg of antibody per ChIP reaction with 25-50μg of chromatin. Include IgG controls and potentially a total H2B antibody control to normalize for histone occupancy. Extended incubation (overnight at 4°C) improves binding efficiency.

Washing and elution:
Implement stringent washing conditions (high salt, LiCl buffers) to reduce background. Elution with 1% SDS at 65°C for 15 minutes followed by reverse cross-linking (65°C for 4 hours or overnight) ensures efficient recovery of bound DNA.

Library preparation and sequencing:
For acetylation marks like H2BK15ac, which may have more diffuse peaks than some other histone modifications, deeper sequencing (30-50 million reads) is recommended to capture the full range of binding sites.

How does Acetyl-HIST1H2BC (K15) occupancy compare with other H2B acetylation marks?

The relationship between different H2B acetylation marks provides important insights into their functional roles and potential coordinated regulation. Research indicates specific patterns of co-occurrence:

Recent studies have shown that H2BK15ac exhibits substantial overlap with other H2B N-terminal acetylation (H2BNTac) sites. ChIP-seq analyses demonstrate that H2BK15ac peaks frequently coincide (approximately 84-96%) with other H2BNTac modifications such as H2BK5ac, H2BK12ac, H2BK16ac, and H2BK20ac . This suggests potential co-regulation of these marks.

The occupancy profile of H2BK15ac most closely resembles that of H2BK16ac and H2BK20ac, while showing distinct patterns from H2BK5ac occupancy . This indicates that different acetylation sites on H2B may serve related but distinct functions.

Compared to H2BK12ac (another well-studied H2B acetylation mark), H2BK15ac typically shows higher peak intensity in ChIP-seq experiments, potentially indicating higher abundance or better antibody enrichment efficiency .

When analyzing genome-wide distributions, H2BK15ac is predominantly found at active regulatory elements, particularly enhancers and promoters of actively transcribed genes, often co-occurring with H3K27ac, a well-established mark of active enhancers .

What are the known writers, readers and erasers of H2B K15 acetylation?

Understanding the enzymes that establish (writers), recognize (readers), and remove (erasers) H2B K15 acetylation is crucial for comprehending its regulatory mechanisms:

Writers (Histone Acetyltransferases):
CBP/p300 represents a primary acetyltransferase complex responsible for H2B K15 acetylation. Studies using the CBP/p300 catalytic inhibitor A-485 have demonstrated significant reduction in H2B K15 acetylation levels . The GCN5/PCAF family of HATs may also contribute to H2B K15 acetylation, particularly in contexts of transcriptional activation.

Readers (Acetylation Recognition Proteins):
Bromodomain-containing proteins, particularly BRD4, have been implicated in recognizing acetylated H2B. The BET family of proteins (including BRD2, BRD3, and BRD4) contain bromodomains that can recognize acetylated lysines on histone tails, including H2B K15ac. The BPTF component of the NURF chromatin remodeling complex contains bromodomains that may recognize H2B K15ac in certain genomic contexts.

Erasers (Histone Deacetylases):
Class I HDACs (HDAC1, HDAC2, HDAC3) can remove acetyl groups from H2B, including K15ac. These enzymes often function within multi-protein complexes such as Sin3A, NuRD, and CoREST. Sirtuins, particularly SIRT1 and SIRT2, may also target H2B K15ac in specific cellular contexts, especially under metabolic stress conditions.

Dynamic regulation through these enzymes allows for rapid changes in H2B K15 acetylation status in response to cellular signals and environmental conditions.

What methodological approaches can resolve antibody specificity issues between similar histone acetylation marks?

Given the challenges with antibody specificity in histone acetylation research, several advanced approaches can help resolve these issues:

Orthogonal validation techniques:
Combining antibody-based detection with mass spectrometry can provide independent verification of the presence and abundance of specific acetylation marks. This approach can help distinguish between similar modifications that might cross-react with antibodies. Systematic peptide array analysis using synthetic peptide arrays containing various acetylated and non-acetylated histone sequences can precisely map antibody specificity and identify potential cross-reactivity .

Genetic approaches:
CRISPR/Cas9-mediated mutation of specific lysine residues (K to R mutations) can eliminate specific acetylation sites, providing definitive controls for antibody specificity testing. These systems allow for direct testing of antibody specificity against specific lysine mutations.

Advanced ChIP methodologies:
CUT&RUN or CUT&Tag techniques offer higher signal-to-noise ratios than traditional ChIP and may provide clearer distinction between similar acetylation marks. Sequential ChIP (re-ChIP) experiments, where material is immunoprecipitated with one antibody followed by a second immunoprecipitation with another antibody, can identify true co-occurrence of modifications versus antibody cross-reactivity.

Computational analysis:
Differential binding analysis comparing ChIP-seq data from multiple antibodies targeting similar modifications can help identify unique and overlapping binding sites. Pattern recognition algorithms can distinguish between true biological signals and technical artifacts due to cross-reactivity .

How should positive and negative controls be designed for Acetyl-HIST1H2BC (K15) antibody validation?

Robust experimental controls are essential for validating the specificity and sensitivity of the Acetyl-HIST1H2BC (K15) antibody:

Positive controls:
Treat cells with histone deacetylase inhibitors (HDACi) such as trichostatin A (TSA) or sodium butyrate to increase global histone acetylation levels, including H2B K15ac. This treatment typically results in enhanced signal when using the antibody in Western blot or immunofluorescence applications .

Use cell lines known to have high levels of H2B K15 acetylation based on published data, such as actively dividing embryonic stem cells or certain cancer cell lines.

Negative controls:
Perform peptide competition assays where the antibody is pre-incubated with excess acetylated H2B K15 peptide before application, which should significantly reduce or eliminate specific signal.

Generate acetylation-deficient mutants by using CRISPR/Cas9 to create K15R mutations in H2B, which prevents acetylation at this site and should result in loss of antibody binding.

Treat cells with CBP/p300 inhibitors like A-485, which should reduce H2B K15 acetylation levels .

Specificity controls:
Include testing against other acetylated lysines on H2B (K5, K12, K16, K20) to confirm the antibody does not cross-react with these sites.

Compare immunoblotting patterns between Acetyl-HIST1H2BC (K15) antibody and pan-H2B antibody to confirm specificity for the modified form.

What are the common pitfalls in Western blot and immunofluorescence when using Acetyl-HIST1H2BC (K15) antibody?

Several technical challenges may arise when using the Acetyl-HIST1H2BC (K15) antibody in Western blot and immunofluorescence applications:

Western blot pitfalls:
Insufficient extraction of nuclear proteins can lead to weak signals. Use RIPA buffer supplemented with protease inhibitors and HDAC inhibitors to prevent deacetylation during extraction.

Standard SDS-PAGE may not provide optimal resolution of histone bands due to their small size. Consider using specialized gel systems designed for histone separation (e.g., Triton-Acid-Urea gels or high percentage (15-18%) SDS-PAGE).

Ensure complete transfer of small histone proteins by using PVDF membranes (rather than nitrocellulose) and adding 0.1% SDS to the transfer buffer.

Non-specific bands may appear due to antibody cross-reactivity. Use appropriate blocking agents (5% BSA is often superior to milk for phospho-specific antibodies) and validate with positive and negative controls.

Immunofluorescence pitfalls:
Inadequate cell fixation can lead to loss of nuclear architecture. Use freshly prepared 4% paraformaldehyde for 10-15 minutes at room temperature.

Insufficient permeabilization may prevent antibody access to nuclear epitopes. After fixation, treat with 0.25-0.5% Triton X-100 for 10 minutes.

High background signals can obscure specific staining. Increase blocking time (1-2 hours) with 5% BSA and include 0.1% Tween-20 in washing steps.

Signal fading during microscopy. Use anti-fade mounting media containing DAPI for nuclear counterstaining and store slides in the dark at 4°C.

For both applications, use the recommended dilutions (WB: 1:500-1:2000; IF: 1:1-1:10) as starting points, but optimize based on your specific experimental conditions and sample types .

How can ChIP-seq data for Acetyl-HIST1H2BC (K15) be analyzed and integrated with other epigenetic marks?

ChIP-seq data analysis for Acetyl-HIST1H2BC (K15) requires specialized approaches for integration with other epigenetic marks:

Basic analysis workflow:
Begin with quality control of sequencing data using FastQC to assess read quality, GC content, and adapter contamination.

Align reads to the reference genome using Bowtie2 or BWA with parameters optimized for histone modification ChIP-seq (allowing for 1-2 mismatches).

Call peaks using MACS2 with parameters appropriate for histone marks (--broad flag for diffuse marks) and using input DNA as control.

Integration with other marks:
Generate heatmaps and aggregation plots centered on transcription start sites, enhancers, or other genomic features using tools like deepTools to visualize the distribution of H2B K15ac relative to these features.

Perform correlation analysis between H2B K15ac and other histone marks (particularly H3K27ac, H3K4me3, and other H2B acetylation marks) to identify patterns of co-occurrence .

Use ChromHMM or similar tools to integrate multiple histone modification datasets and define chromatin states across the genome.

Functional analysis:
Associate H2B K15ac peaks with genes using tools like GREAT or by proximity to gene promoters and correlate with gene expression data from RNA-seq.

Perform motif enrichment analysis on H2B K15ac peaks to identify potential transcription factors associated with this mark.

Conduct pathway analysis of genes associated with H2B K15ac to identify biological processes regulated by this modification.

Visualization and sharing:
Create genome browser tracks using IGV or the UCSC Genome Browser to visualize H2B K15ac distribution along with other genomic features.

Consider depositing processed data in repositories like GEO or ArrayExpress with appropriate metadata for reproducibility.

What factors influence the variability in Acetyl-HIST1H2BC (K15) detection across different experimental systems?

Several factors can contribute to variability in detecting Acetyl-HIST1H2BC (K15) across different experimental systems:

Biological factors:
Cell type-specific differences in H2B variant expression and acetylation patterns can significantly impact detection levels. Embryonic stem cells often show higher levels of active histone marks compared to differentiated cells.

Cell cycle stage affects histone acetylation patterns, with levels typically higher during S-phase when chromatin assembly occurs.

Cellular stress responses (oxidative stress, heat shock, nutrient deprivation) can rapidly alter histone acetylation states through changes in HAT and HDAC activities.

Technical factors:
Antibody lot-to-lot variability can significantly impact detection consistency. Always validate new lots against previous ones and consider creating a reference standard.

Sample preparation methods, particularly the presence or absence of HDAC inhibitors during extraction, can affect acetylation levels. Include sodium butyrate or other HDAC inhibitors in all buffers to prevent deacetylation during processing.

Fixation conditions for ChIP or IF (duration, crosslinker concentration, temperature) can affect epitope accessibility. Optimize for each cell type and application.

Storage conditions of samples and antibodies influence stability of both the modification and the detection reagent. Avoid repeated freeze-thaw cycles and store antibodies according to manufacturer recommendations.

Analytical factors:
Normalization methods in quantitative analyses can affect interpretation of results. For ChIP-seq, normalize to input DNA and consider spike-in controls for cross-sample comparisons.

Image acquisition parameters for IF (exposure time, gain settings) must be standardized across experiments for valid comparisons.

How can Acetyl-HIST1H2BC (K15) antibodies be used to study the interplay between histone modifications and DNA damage response?

The Acetyl-HIST1H2BC (K15) antibody provides valuable insights into the relationship between histone modifications and DNA damage response (DDR):

Experimental approaches:
Time-course ChIP experiments following DNA damage induction (with agents like etoposide, neocarzinostatin, or UV irradiation) can reveal dynamic changes in H2B K15 acetylation patterns at damage sites and globally. Combine with γH2AX ChIP to correlate H2B K15ac changes with DNA damage markers.

Immunofluorescence co-localization studies can visualize the spatial relationship between H2B K15ac and DDR proteins like 53BP1, BRCA1, or RAD51 at sites of damage. This can help establish whether H2B K15ac is recruited to or excluded from damage sites.

CRISPR/Cas9-mediated mutation of H2B K15 (K15R to prevent acetylation) followed by DNA damage sensitivity assays can determine the functional importance of this modification in DNA repair processes.

Mechanistic investigations:
Investigate the recruitment of HATs like CBP/p300 to DNA damage sites and their role in modulating H2B K15 acetylation during repair.

Examine how H2B K15 acetylation affects chromatin accessibility at damage sites using techniques like ATAC-seq in conjunction with ChIP-seq.

Explore the interplay between H2B K15ac and other damage-responsive histone modifications (such as γH2AX, H4K16ac, H3K9me3) during different phases of the DNA damage response.

This research direction might reveal novel roles for H2B K15 acetylation in facilitating DNA repair processes through chromatin structure modulation.

What approaches can be used to study the dynamics of Acetyl-HIST1H2BC (K15) during cell differentiation and development?

Studying the dynamics of Acetyl-HIST1H2BC (K15) during cellular differentiation provides insights into epigenetic reprogramming mechanisms:

Temporal profiling methodologies:
Perform time-course ChIP-seq experiments during directed differentiation of stem cells (e.g., embryonic stem cells to specific lineages) to map changes in H2B K15ac genome-wide. Integrate with transcriptome data to correlate acetylation changes with gene expression dynamics.

Single-cell techniques like scCUT&Tag can capture heterogeneity in H2B K15ac patterns during differentiation at the individual cell level, potentially identifying epigenetic changes preceding transcriptional decisions.

CUT&RUN or CUT&Tag approaches provide higher resolution with lower background than traditional ChIP and can be performed with fewer cells, making them suitable for limited developmental samples.

Functional validation approaches:
CRISPR/Cas9-mediated interference with writers (HATs) or erasers (HDACs) of H2B K15ac can test the functional requirement of this modification during differentiation.

Use of small molecule inhibitors of HATs or HDACs at specific time windows during differentiation can reveal critical periods when H2B K15ac dynamics are essential for proper developmental progression.

H2B K15R mutant expression (preventing acetylation) can be used to assess the requirement for this specific modification in differentiation models.

Integrative analysis:
Combining H2B K15ac profiles with other epigenetic marks (H3K4me3, H3K27me3, H3K27ac) can identify bivalent domains and regions undergoing epigenetic transitions during development.

Integration with transcription factor binding data can reveal potential regulatory relationships and identify factors that may recruit or be recruited by H2B K15ac during cell fate decisions.

Computational modeling of the temporal relationship between H2B K15ac changes and gene expression can help establish causality in epigenetic regulation during development.

How can mass spectrometry complement antibody-based detection of Acetyl-HIST1H2BC (K15)?

Mass spectrometry (MS) provides complementary and often more definitive information about histone modifications compared to antibody-based approaches:

Advantages of MS approaches:
MS can simultaneously detect multiple histone modifications, allowing for comprehensive profiling of the "histone code" including combinations of marks that may be functionally important.

MS provides absolute quantification of modification abundance, overcoming the semi-quantitative nature of antibody-based methods.

MS can distinguish between modifications with similar molecular weights (e.g., acetylation vs. trimethylation) that may be challenging for antibodies to differentiate.

Experimental workflows:
For bottom-up histone MS, extract histones using acid extraction, perform propionylation to block unmodified lysines, digest with trypsin, and analyze peptides by LC-MS/MS. This approach can quantify H2B K15ac along with other modifications.

For middle-down approaches, use GluC instead of trypsin to generate larger histone fragments that preserve combinatorial modification patterns on the same histone tail.

For top-down analysis, analyze intact histone proteins to preserve all modifications and their combinations, though this requires specialized high-resolution MS equipment.

Integration with antibody-based methods:
Use MS to validate antibody specificity by analyzing immunoprecipitated material from ChIP experiments with the Acetyl-HIST1H2BC (K15) antibody.

Employ MS to identify novel modification combinations involving H2B K15ac that may not be detectable by available antibodies.

Correlate MS-based quantification of H2B K15ac with ChIP-seq or IF intensity measurements to calibrate antibody-based approaches.

Apply stable isotope labeling (SILAC or similar approaches) to quantitatively track H2B K15ac dynamics during cellular processes with greater precision than is possible with antibody methods alone.

What are the emerging technologies that will advance research on Acetyl-HIST1H2BC (K15)?

Several cutting-edge technologies are poised to transform research on Acetyl-HIST1H2BC (K15) and related histone modifications:

Single-molecule approaches:
Single-molecule imaging techniques using super-resolution microscopy (like STORM or PALM) combined with specific Acetyl-HIST1H2BC (K15) antibodies can visualize the spatial distribution of this modification at unprecedented resolution.

Single-cell epigenomic profiling methods (scCUT&Tag, scATAC-seq) will reveal cell-to-cell variation in H2B K15ac distribution, particularly valuable in heterogeneous tissues or during developmental transitions.

CRISPR-based tools:
CRISPR activation/repression systems fused to histone-modifying enzymes can enable site-specific manipulation of H2B K15ac at specific genomic loci, allowing causal testing of this modification's function.

Base editing approaches could enable conversion of lysine to other amino acids, providing more precise tools to study the function of H2B K15 acetylation than traditional mutagenesis.

Computational advances:
Machine learning algorithms trained on integrated multi-omics datasets will improve prediction of functional consequences of H2B K15ac patterns across the genome.

Network analysis approaches will better define the relationship between H2B K15ac and other chromatin features in determining gene expression outcomes.

Therapeutic applications:
Development of small molecules specifically targeting the writers, readers, or erasers of H2B K15ac could provide new therapeutic approaches for diseases with epigenetic dysregulation.

These technological advances will not only deepen our understanding of H2B K15 acetylation biology but may also reveal new therapeutic targets and diagnostic approaches based on this epigenetic modification.

How might current research on Acetyl-HIST1H2BC (K15) translate to clinical applications?

The study of Acetyl-HIST1H2BC (K15) has several potential clinical applications and therapeutic implications:

Diagnostic applications:
Changes in H2B K15ac patterns may serve as biomarkers for certain diseases, particularly cancers where epigenetic dysregulation is common. Antibody-based detection of this modification in patient samples could aid in diagnosis or prognosis.

Integration of H2B K15ac patterns with other epigenetic marks could generate "epigenetic signatures" with potential diagnostic value for conditions ranging from cancer to neurodevelopmental disorders.

Therapeutic strategies:
Modulation of H2B K15ac through targeting of specific HATs (like CBP/p300) or HDACs that regulate this modification could represent a novel therapeutic approach. Several HDAC inhibitors are already approved for cancer treatment, and more specific agents could be developed.

Understanding the relationship between H2B K15ac and cellular differentiation could inform regenerative medicine approaches, potentially allowing for more efficient directed differentiation protocols for stem cell therapies.

Precision medicine applications:
Characterization of individual patients' H2B K15ac patterns might help predict response to epigenetic therapies, enabling more personalized treatment approaches.

Combining H2B K15ac profiling with genetic information could identify patient subgroups most likely to benefit from specific interventions targeting epigenetic mechanisms.