HIST1H3A (Ab-27) Antibody

What is the HIST1H3A (Ab-27) antibody and what epitope does it specifically recognize?

The HIST1H3A (Ab-27) antibody is a polyclonal antibody generated in rabbits that specifically recognizes the dimethylated lysine 27 (H3K27me2) on histone H3. The immunogen used for generating this antibody is a synthetic peptide sequence around the di-methylation site of lysine 27 (A-R-K(di-methyl)-S-A) derived from Human Histone H3 . This antibody is particularly valuable for studying epigenetic modifications as H3K27me2 represents an important repressive chromatin mark that regulates gene expression during development and cellular differentiation. The antibody has been purified by affinity chromatography using epitope-specific phosphopeptides, with non-phospho specific antibodies removed through chromatography to ensure high specificity .

What applications has the HIST1H3A (Ab-27) antibody been validated for?

The HIST1H3A (Ab-27) antibody has been validated for multiple research applications, enabling comprehensive epigenetic studies:

The antibody is supplied in an unconjugated form, making it versatile for various detection methods depending on the experimental setup . For specialized applications like ChIP (Chromatin Immunoprecipitation), preliminary optimization is recommended as conditions may vary based on target cell type and experimental conditions.

What is known about the cross-reactivity profile of the HIST1H3A (Ab-27) antibody?

The HIST1H3A (Ab-27) antibody exhibits cross-reactivity across multiple species, making it valuable for comparative studies:

This multi-species reactivity is particularly useful for evolutionary studies of epigenetic modifications and allows researchers to maintain consistent reagents across different model systems . When using this antibody in untested species or cell types, proper validation controls should be included to confirm specific binding to H3K27me2.

What are the optimal protocols for using HIST1H3A (Ab-27) antibody in chromatin immunoprecipitation (ChIP) experiments?

When designing ChIP experiments with the HIST1H3A (Ab-27) antibody, researchers should follow these methodological guidelines for optimal results:

• Crosslinking and Chromatin Preparation:

  • Use 1% formaldehyde for 10 minutes at room temperature for protein-DNA crosslinking

  • Sonicate chromatin to generate fragments of 200-500 bp (verify fragment size by gel electrophoresis)

  • Pre-clear chromatin with protein A/G beads to reduce non-specific binding

• Immunoprecipitation:

  • Use 3-5 μg of HIST1H3A (Ab-27) antibody per 25-50 μg of chromatin

  • Include appropriate controls: IgG negative control, input sample (10% of starting material), and a positive control antibody (total H3)

  • Incubate overnight at 4°C with gentle rotation

• Washing and Elution:

  • Perform stringent washes with increasing salt concentrations to reduce background

  • Elute immunoprecipitated material with SDS-containing buffer at 65°C

  • Reverse crosslinks (65°C overnight) and treat with proteinase K

• Analysis Considerations:

  • H3K27me2 often shows broader distribution patterns compared to H3K27me3

  • Include known H3K27me2-enriched regions as positive controls in qPCR validation

  • For genome-wide analysis, H3K27me2 ChIP-seq typically requires deeper sequencing compared to H3K27me3 ChIP-seq

Understanding the relationship between H3K27me2 and gene regulation requires careful experimental design, as H3K27me2 can serve as an intermediate state between active chromatin and the more repressive H3K27me3 modification .

How should researchers interpret H3K27me2 patterns in relation to gene expression data?

Interpreting the relationship between H3K27me2 patterns and gene expression requires understanding several key principles:

• Regulatory Context:

  • H3K27me2 generally correlates with transcriptional repression but shows more nuanced patterns than H3K27me3

  • H3K27me2 often marks gene bodies and intergenic regions, while H3K27me3 is concentrated at promoters

  • The relationship between H3K27me2 and expression varies by genomic context and cell type

• Developmental Transitions:

  • H3K27me2 can represent an intermediate state in gene silencing or activation

  • During differentiation, genes may transition through different H3K27 methylation states (unmodified → me1 → me2 → me3 or in reverse)

  • Time-course experiments during development reveal dynamic relationships between methylation states and gene activation

• Co-occurrence with Other Modifications:

  • H3K27me2 effects are modulated by co-occurring modifications

  • H3K27ac is antagonistic to methylation and marks active enhancers

  • Regional analysis of multiple modifications provides better predictive power for gene expression

• Data Integration Approach:

Studies in both plant and animal models have shown that H3K27 modifications play critical roles in developmental gene regulation, with mutations at this residue causing severe developmental phenotypes .

What controls and validation steps are essential when using HIST1H3A (Ab-27) antibody?

Rigorous validation of the HIST1H3A (Ab-27) antibody is crucial for reliable research outcomes:

• Specificity Controls:

  • Peptide competition assays using H3K27me2 peptides to confirm specific binding

  • Parallel testing with other validated H3K27me2 antibodies to compare signal patterns

  • Testing on modified peptide arrays to assess cross-reactivity with other methylation states (H3K27me1/me3)

• Biological Validation:

  • Use of EZH1/2 inhibitors or knockdowns to reduce H3K27me2 levels as negative controls

  • Testing in cell lines with known H3K27me2 patterns as positive controls

  • Include isotype control antibodies to assess non-specific binding

• Technical Validation:

  • Titration experiments to determine optimal antibody concentration

  • Inclusion of recombinant histone standards with defined modification states

  • Western blot validation showing single band at approximately 17 kDa

• Application-Specific Validation:

  • For IHC/IF: Include tissues with known H3K27me2 patterns and test multiple fixation methods

  • For ChIP: Validate enrichment at known H3K27me2-positive regions by qPCR before sequencing

  • For mass spectrometry studies: Confirm pull-down specificity by analyzing modification state of immunoprecipitated histones

Proper validation is particularly important as H3K27me2 patterns can vary significantly between normal and disease states, as seen in studies of DICER1-associated tumors which show characteristic losses of H3K27me3 immunostaining .

How can HIST1H3A (Ab-27) antibody be utilized to investigate the dynamics between different H3K27 methylation states?

The HIST1H3A (Ab-27) antibody enables sophisticated studies of H3K27 methylation dynamics:

• Sequential ChIP (Re-ChIP) Approaches:

  • First IP with HIST1H3A (Ab-27) antibody followed by second IP with anti-H3K27me3 antibody

  • Reveals regions undergoing transition between methylation states

  • Identifies genomic loci with heterogeneous nucleosome populations

• Time-Course Analysis During Cell Differentiation:

  • Track H3K27me2 levels at key developmental genes during differentiation

  • Correlate changes in methylation state with transcriptional activation/repression

  • Map the temporal order of epigenetic changes preceding gene expression changes

• Enzyme Inhibitor Studies:

  • Monitor H3K27me2 redistribution after treatment with EZH2 inhibitors

  • Study the conversion between methylation states during drug treatment

  • Correlate with changes in gene expression for therapeutic target identification

• Integrated Multi-Omic Profiling:

  • Combined analysis of H3K27me2, H3K27me3, and H3K27ac distributions

  • Integration with chromatin accessibility data (ATAC-seq)

  • Creation of comprehensive epigenetic landscapes during development or disease progression

Research has demonstrated that specific H3K27 methylation states on histone variant H3.3 play distinct roles in regulating lineage-specific genes and terminal differentiation programs , highlighting the importance of studying specific histone variants and their modifications.

The HIST1H3A (Ab-27) antibody recognizes H3K27me2 across different H3 variants, enabling comparative studies:

• Histone Variant-Specific Functions:

  • H3.3K27 methylation plays distinct roles from canonical H3K27 methylation

  • H3.3 is deposited in a replication-independent manner at active genes and regulatory elements

  • Studies in Drosophila show that H3.3K27 is required for proper development

• Technical Considerations for Variant-Specific Analysis:

  • ChIP-seq with variant-specific antibodies reveals distinct genomic distributions

  • Mass spectrometry approaches can quantify modification levels on specific variants

  • Genetic approaches using K27 mutations in H3.3 genes reveal variant-specific functions

• Research Findings on Variant-Specific Roles:

  • In Arabidopsis, H3.3K27A variants cause severe developmental defects, demonstrating crucial roles in plant cell fates and metabolic pathways

  • H3.3K27me3 shows unique enrichment at lineage-specific genes in mouse embryonic stem cells

  • H3.3K27 modifications regulate distinct terminal differentiation genes compared to canonical H3K27 modifications

• Experimental Approaches:

  • Custom antibodies specific for variant-H3K27me2 enable more precise studies

  • CRISPR/Cas9-mediated mutation of K27 in H3.3-encoding genes (as done in Drosophila and Arabidopsis models)

  • Multi-omic profiling comparing canonical and variant histone modifications

Research has demonstrated that "while canonical H3K27me3 has been characterized to regulate the expression of transcription factors that play a general role in differentiation, H3.3K27me3 is essential for regulating distinct terminal differentiation genes" .

What are common sources of false positives/negatives when using HIST1H3A (Ab-27) antibody and how can they be addressed?

When using HIST1H3A (Ab-27) antibody, researchers should be aware of these potential artifacts:

• Common Sources of False Positives:

  • Cross-reactivity with H3K27me1 or H3K27me3 modifications

  • Non-specific binding to other methylated lysine residues

  • Inadequate blocking leading to high background in immunostaining

  • Fixation artifacts in tissue samples causing epitope masking

• Common Sources of False Negatives:

  • Over-fixation leading to epitope masking

  • Insufficient antigen retrieval in formalin-fixed tissues

  • Degradation of modifications during sample preparation

  • Competitive binding from other proteins in the nuclear environment

• Troubleshooting Strategies:

• Validation Approaches:

  • Include parallel staining with total H3 antibody as control for histone accessibility

  • Use competing peptides to confirm signal specificity

  • Include biological controls with known H3K27me2 status

  • Confirm key findings with orthogonal methods (e.g., mass spectrometry)

Proper experimental design and controls are especially important when studying diseases with altered H3K27 methylation patterns, such as DICER1-associated tumors which show characteristic loss of H3K27me3 .

How should researchers integrate H3K27me2 data with other epigenetic marks for comprehensive chromatin state analysis?

Integrative analysis approaches for H3K27me2 data include:

• Multi-Mark Chromatin State Modeling:

  • Combine H3K27me2 with other histone modifications in computational frameworks

  • Employ machine learning algorithms to define chromatin states

  • Integrate with DNA methylation and chromatin accessibility data

• Hierarchical Analysis Framework:

• Biological Context Integration:

  • Correlate H3K27me2 patterns with developmental trajectories

  • Map changes during cell fate transitions

  • Compare normal versus disease states to identify pathological alterations

• Visualization and Analysis Tools:

  • Genome browsers for multi-track visualization

  • Heatmaps showing mark co-occurrence at genomic features

  • Metaplot analysis around transcription start sites and enhancers

Studies in Arabidopsis have shown how integrating H3K27 methylation data with transcriptomics and metabolomics can reveal novel roles in plant development and lignin biosynthesis , demonstrating the power of multi-omic approaches.

What special considerations apply when using HIST1H3A (Ab-27) antibody for diagnostic or clinical research applications?

For diagnostic and clinical applications, researchers should consider:

• Standardization Requirements:

  • Establish standardized protocols for sample processing

  • Use automated staining platforms when possible

  • Develop quantitative scoring systems for H3K27me2 levels

• Tissue-Specific Considerations:

  • Optimize fixation protocols for each tissue type

  • Determine appropriate antigen retrieval methods

  • Establish tissue-specific positive and negative controls

• Diagnostic Value Assessment:

  • Compare H3K27me2 patterns with established diagnostic markers

  • Correlate patterns with clinical outcomes in retrospective studies

  • Evaluate sensitivity and specificity for specific disease detection

• Clinical Sample Challenges:

• Emerging Applications:

  • Detection of H3K27me2/me3 loss in specific tumor types

  • Monitoring epigenetic changes during treatment

  • Identifying patients likely to respond to epigenetic therapies

Research has established that H3K27me3 loss can serve as a helpful diagnostic marker for certain tumors like DICER1-associated primary intracranial sarcomas and PPB types II and III , suggesting potential diagnostic applications for H3K27 methylation profiling.

What emerging technologies might enhance H3K27me2 research beyond current antibody-based methods?

Future research on H3K27me2 will benefit from these emerging technologies:

• Single-Cell Epigenomic Approaches:

  • Single-cell CUT&Tag for H3K27me2 profiling with cellular resolution

  • Integrated single-cell multi-omic methods (scNOMe-seq, scCUT&Tag)

  • Computational methods for trajectory analysis of epigenetic states

• Advanced Imaging Techniques:

  • Super-resolution microscopy of H3K27me2 distribution

  • Live-cell imaging using engineered readers for H3K27me2

  • Multiplexed imaging of multiple histone modifications

• Direct Modification Detection Methods:

  • Third-generation sequencing for direct detection of modifications

  • Mass spectrometry approaches with improved sensitivity

  • Antibody-free detection using engineered readers or chemical approaches

• Variant-Specific Analysis:

  • Techniques to distinguish modifications on specific H3 variants

  • Custom antibodies for variant-specific H3K27me2 detection

  • Computational methods to resolve variant-specific signals from sequencing data

This continues to be an evolving field, with recent studies demonstrating unique roles for H3.3K27 methylation in regulating lineage-specific genes and terminal differentiation programs .

What are the most pressing unanswered questions regarding H3K27me2 biology?

Critical questions in H3K27me2 research include:

• Regulatory Mechanisms:

  • How is the balance between H3K27me2 and other methylation states dynamically regulated?

  • What determines the specificity of H3K27 methyltransferases for me1, me2, or me3 states?

  • What are the specific readers of H3K27me2 distinct from H3K27me3 readers?

• Functional Roles:

  • What is the distinct function of H3K27me2 compared to H3K27me3?

  • How does H3K27me2 on histone variants like H3.3 differ functionally from canonical H3?

  • What role does H3K27me2 play in enhancer regulation and 3D genome organization?

• Disease Relevance:

  • How are H3K27me2 patterns specifically altered in different disease contexts?

  • Can H3K27me2 patterns serve as prognostic or predictive biomarkers?

  • How do mutations in epigenetic regulators specifically impact H3K27me2 vs. H3K27me3?

• Developmental Biology:

  • What is the role of H3K27me2 in cell fate decisions during development?

  • How do H3K27me2 patterns evolve during cellular differentiation?

  • What is the evolutionary conservation of H3K27me2 functions across species?

Recent research in Arabidopsis and Drosophila has begun addressing some of these questions, revealing critical roles for H3K27 modifications in development and gene regulation .

How might advances in computational approaches enhance the interpretation of H3K27me2 data?

Computational advances for H3K27me2 research include:

• Advanced Peak Calling Algorithms:

  • Methods optimized for broad domains characteristic of H3K27me2

  • Differential binding analysis accounting for variability in broad marks

  • Integration of multiple data types for improved signal detection

• Machine Learning Applications:

  • Classification of chromatin states incorporating H3K27me2

  • Predictive models for gene expression based on histone modification patterns

  • Transfer learning approaches leveraging data across cell types and conditions

• Network-Based Analyses:

  • Construction of epigenetic regulatory networks

  • Integration of epigenetic data with transcription factor binding networks

  • Systems biology approaches to model modification dynamics

• Multi-Omics Integration:

  • Methods to correlate H3K27me2 patterns with transcriptome, proteome, and metabolome

  • Causal inference approaches to determine regulatory relationships

  • Multi-modal data visualization and exploration tools

Integrative computational approaches have already revealed important insights, such as the role of H3.3K27me3 in regulating distinct terminal differentiation genes compared to canonical H3K27me3 .