H3F3A Antibody Pair

What is H3F3A and why are specific antibodies needed for its research?

H3F3A encodes the histone H3.3 protein variant (15.3 kDa), a replication-independent histone that plays critical roles in chromatin organization and gene regulation. Specific antibodies are essential for H3F3A research for several reasons:

• Genetic distinction: Though H3F3A and H3F3B encode identical proteins, they have different genomic locations (chromosomes 1 and 17, respectively) and distinct regulatory mechanisms, requiring antibodies that can distinguish their unique expression patterns .

• Cancer-specific mutations: H3F3A harbors recurrent mutations (K27M, G34R/V/W) that drive specific tumor types, necessitating mutation-specific antibodies for accurate tumor classification and mechanistic studies .

• Post-translational modification detection: Antibodies recognizing specific modifications (phosphorylation at Thr3, acetylation at Lys36, methylation at K27/K36) enable researchers to study how these modifications alter histone function .

• Subcellular localization: Antibodies facilitate visualization of H3F3A distribution in cellular compartments, particularly within chromatin, using immunohistochemistry and immunofluorescence techniques .

For optimal experimental design, researchers should consider whether their question requires antibodies against wild-type H3.3, specific mutations, or particular post-translational modifications, as each provides different biological insights.

How can researchers distinguish between H3F3A and H3F3B mutations using antibodies?

Distinguishing between mutations in H3F3A and H3F3B requires sophisticated antibody selection and validation strategies:

• Mutation-specific antibodies: Commercial antibodies are available that specifically recognize the K27M, G34R, or G34V mutations in histone H3.3. These antibodies are developed against synthetic peptides containing the mutated sequence and show high sensitivity and specificity for their targets .

• Validation protocols: Researchers should implement rigorous validation using:

  • Western blotting against recombinant wild-type and mutant proteins

  • Immunohistochemistry on known positive and negative tissues

  • Peptide competition assays to confirm epitope specificity

  • Cross-validation with gene sequencing data

• Tumor-specific patterns: Understanding the typical mutation patterns can aid interpretation:

This remarkable tumor specificity helps in designing control experiments and interpreting antibody staining patterns in diagnostic applications .

What technical considerations are essential when selecting H3F3A antibodies for specific experimental applications?

Selecting appropriate H3F3A antibodies requires careful consideration of multiple technical parameters:

• Antibody specificity: Determine whether you need an antibody that recognizes:

  • Wild-type H3F3A protein

  • Specific mutations (K27M, G34R/V)

  • Post-translational modifications (e.g., phosphorylation at Thr3)

• Clonality selection:

  • Monoclonal antibodies: Offer higher specificity but potentially lower sensitivity

  • Polyclonal antibodies: Often provide higher sensitivity but may have increased cross-reactivity

  • Recombinant monoclonal antibodies: Combine specificity with batch-to-batch consistency

• Application optimization:

  • Western blot: 1:500-2000 dilution; use high percentage (15-18%) gels

  • IHC: 1:100-300 dilution; require robust antigen retrieval protocols

  • ICC/IF: 1:200-1000 dilution; permeabilization optimization critical

  • ChIP: Specific antibodies validated for chromatin binding

• Species reactivity: Verify cross-reactivity with your model organism (human, mouse, rat)

• Conjugation requirements: Consider whether unconjugated antibodies are sufficient or if fluorophore/enzyme conjugates (PE, FITC, HRP) are needed for your detection system

The search results indicate over 1,500 commercially available H3F3A antibodies across 38 suppliers, highlighting the importance of careful selection based on experimental needs .

How can researchers use H3F3A mutation-specific antibodies to study cancer mechanisms?

H3F3A mutation-specific antibodies provide powerful tools for investigating oncogenic mechanisms through several methodological approaches:

• Tumor classification: Mutation-specific antibodies enable precise classification of tumors based on their histone mutation status, which has significant prognostic implications. For example, H3.3K27M antibodies help identify diffuse midline gliomas with poor prognosis .

• Mechanistic pathway studies: These antibodies facilitate elucidation of downstream effector pathways. For instance, H3.3K27M overexpression has been shown to increase levels of β-catenin, p-β-catenin (Ser675), USP1, and EZH2, promoting glioma cell migration and invasion through the β-catenin/USP1/EZH2 pathway .

• Functional validation methodology:

  • Generate stable cell lines expressing mutant H3.3 proteins

  • Confirm expression using mutation-specific antibodies via Western blot

  • Conduct phenotypic assays (proliferation, migration, invasion)

  • Analyze signaling pathway activation using phospho-specific antibodies

  • Verify mutation effects using inhibitors of identified pathways

• Chromatin dynamics: Chromatin immunoprecipitation (ChIP) with H3F3A mutation-specific antibodies enables mapping of mutant histone distribution and associated chromatin modifications. This approach has revealed that RACK7 (ZMYND8) recognizes H3.3G34R mutation to suppress expression of specific genes, including CIITA .

• In vivo tumor models: Immunohistochemical analysis using mutation-specific antibodies can validate the expression and effects of histone mutations in xenograft or genetically engineered mouse models .

This multilayered approach provides comprehensive insights into how specific histone mutations drive tumorigenesis through alterations in chromatin structure and gene expression.

What validation methods ensure specificity of H3F3A mutation-specific antibodies?

Rigorous validation of H3F3A mutation-specific antibodies is essential for research integrity. A comprehensive validation protocol should include:

• Genetic controls:

  • Test antibodies on cell lines with sequencing-verified mutation status

  • Use CRISPR-Cas9 engineered cell lines expressing specific mutations

  • Include wild-type controls to confirm absence of cross-reactivity

• Peptide competition assays: Pre-incubate antibodies with synthetic peptides containing:

  • The specific mutation (should eliminate signal)

  • Wild-type sequence (should not affect binding)

  • Related mutations (to test cross-reactivity)

• Multi-technique validation: Confirm specificity across:

  • Western blot

  • Immunohistochemistry

  • Immunofluorescence

  • Flow cytometry

  This cross-platform validation is critical as epitope accessibility varies between techniques .

• Dilution optimization: Establish optimal signal-to-noise ratio through serial dilutions (typically 1:100-1:2000 depending on application) .

• DNA sequencing correlation: Validate antibody results against direct DNA sequencing of the H3F3A gene using primers such as:

  • H3F3A-F: 5'-ATG GCT CGT ACA AAG CAG AC-3'

  • H3F3A-R: 5'-AGC ACG TTC TCC ACG TAT GC-3'

As demonstrated in the literature, H3.3K27M antibodies have been validated on human glioma tissues with confirmed AAG→ATG codon mutations at position 27, confirming their specificity and utility for diagnostic applications .

How do post-translational modifications affect H3F3A antibody recognition?

Post-translational modifications (PTMs) significantly impact H3F3A antibody recognition through multiple mechanisms that researchers must account for in experimental design:

• Epitope masking effects: PTMs can alter histone three-dimensional structure, potentially concealing epitopes. For instance, phosphorylation of Ser31 might affect accessibility of nearby Lys36, although the Anti-Acetyl-Histone H3 (Lys36) antibody maintains specificity despite neighboring modifications .

• Modification-specific recognition: Many antibodies are specifically designed to recognize certain PTMs, including:

  • Phosphorylation at Thr3 - recognized by Rabbit anti Human histone H3F3A (pThr3) antibody

  • Acetylation at Lys36 - detected by Anti-Acetyl-Histone H3 (Lys36) antibody

  • Methylation states (mono-, di-, or tri-) at Lys36 or Lys27

• Cross-talk between modifications: Research has demonstrated that some modifications influence the recognition of nearby epitopes. For example, studies on H3.3G34R binding to RACK7 showed that:

  • H3K14 acetylation did not influence G34R binding to RACK7

  • Mono-, di-, and trimethylation of H3.3K36 did not interfere with G34R-RACK7 interaction

• Sample preparation considerations: Different extraction methods can preserve or disrupt specific PTMs:

  • Acid extraction enhances histone recovery but may alter some PTMs

  • Use of phosphatase inhibitors is critical when studying phosphorylation

  • Deacetylase inhibitors should be included when examining acetylation marks

• Antibody selection strategy: When studying PTMs, researchers should select antibodies raised against the specific modification of interest, with validation data demonstrating both the recognition of the modified residue and the lack of cross-reactivity with the unmodified form or other similar modifications .

Understanding these complex interactions is essential for proper experimental design and interpretation when studying the diverse epigenetic roles of H3F3A.

Western Blot Protocol for H3F3A Detection

• Sample preparation:

  • Extract histones using acid extraction (0.2N HCl) to enrich for basic proteins

  • Include protease inhibitors and phosphatase inhibitors (for phospho-epitopes)

  • Quantify protein concentration using Bradford or BCA assay

• Gel electrophoresis:

  • Use 15-18% SDS-PAGE to effectively resolve the small (15.3 kDa) H3F3A protein

  • Load 10-20 μg of total protein or 2-5 μg of purified histones

  • Run at 100-120V until dye front reaches bottom

• Transfer and detection:

  • Transfer to PVDF membrane (100V for 1 hour or 30V overnight)

  • Block with 5% BSA in TBST (not milk, which contains phosphatases)

  • Incubate with primary antibody at 1:500-2000 dilution overnight at 4°C

  • Wash 5× with TBST, 5 minutes each

  • Apply HRP-conjugated secondary antibody at 1:5000-10000 for 1 hour

  • Develop using enhanced chemiluminescence

• Controls:

  • Include positive control (cell line with known H3F3A expression)

  • For mutation-specific antibodies, include both mutant and wild-type samples

Immunohistochemistry Protocol

• Tissue preparation:

  • Fix tissues in 10% neutral buffered formalin (12-24 hours)

  • Process, embed in paraffin, and section at 4-5 μm thickness

• Staining procedure:

  • Deparaffinize and rehydrate sections

  • Perform heat-induced epitope retrieval using citrate buffer (pH 6.0)

  • Block endogenous peroxidase with 3% H₂O₂, 10 minutes

  • Block with 5-10% normal serum, 1 hour

  • Apply primary antibody at 1:100-300 dilution, overnight at 4°C

  • Apply appropriate detection system (HRP-polymer recommended)

  • Develop with DAB substrate, counterstain with hematoxylin

  • Dehydrate and mount

• Optimization considerations:

  • Antigen retrieval method is critical for histone epitopes

  • Dilution optimization should be performed for each new antibody lot

  • Include known positive and negative controls

Chromatin Immunoprecipitation (ChIP)

• Chromatin preparation:

  • Crosslink cells with 1% formaldehyde, 10 minutes at room temperature

  • Quench with 125 mM glycine, 5 minutes

  • Lyse cells and sonicate to generate 200-500 bp fragments

  • Check sonication efficiency by agarose gel electrophoresis

• Immunoprecipitation:

  • Pre-clear chromatin with protein A/G beads

  • Incubate chromatin with H3F3A antibody (2-5 μg per reaction) overnight

  • Add protein A/G beads, incubate 2-3 hours

  • Wash extensively with increasingly stringent buffers

  • Elute, reverse crosslinks, and purify DNA

• Analysis options:

  • qPCR for specific genomic regions

  • ChIP-seq for genome-wide profiling

  • Re-ChIP for sequential immunoprecipitation with different antibodies

These protocols provide starting points that should be optimized for specific antibodies and experimental conditions.

How can ChIP assays with H3F3A antibodies provide insights into chromatin dynamics?

Chromatin Immunoprecipitation (ChIP) assays with H3F3A antibodies offer powerful methodological approaches to investigate chromatin structure and function:

• Genome-wide distribution mapping: ChIP-seq with H3F3A antibodies reveals the genomic localization patterns of histone H3.3, which is typically enriched at:

  • Actively transcribed genes

  • Promoters and enhancers

  • Sites of DNA repair

  • Telomeres and centromeres

• Mutation impact analysis: Using mutation-specific antibodies (e.g., H3.3K27M, H3.3G34R) in ChIP-seq allows researchers to determine how these oncogenic mutations alter:

  • Histone deposition patterns

  • Associated epigenetic modifications

  • Recruitment of chromatin modifiers

  • Transcriptional output

  For example, researchers demonstrated that H3.3G34R is enriched at specific genomic loci including CIITA, GFAP, and other genes in pediatric glioblastoma .

• Integration with epigenomic techniques: ChIP data from H3F3A antibodies can be integrated with:

  • RNA-seq for correlation with transcriptional output

  • ATAC-seq for chromatin accessibility analysis

  • DNA methylation profiling for comprehensive epigenetic landscapes

  • Hi-C for three-dimensional chromatin organization

• Protocol optimization for histone ChIP:

  • Use low-cell number protocols for precious samples (tumor biopsies)

  • Optimize sonication conditions for consistent chromatin fragmentation

  • Include spike-in controls for quantitative comparisons

  • Perform sequential ChIP to analyze co-occurrence of different modifications

• Data analysis considerations:

  • Use appropriate peak-calling algorithms for histone modifications

  • Perform differential binding analysis between conditions

  • Consider nucleosome positioning in data interpretation

  • Correlate with published datasets on histone variant dynamics

These approaches have revealed critical insights, such as how H3.3G34R mutation affects gene expression through interaction with the chromatin reader RACK7 (ZMYND8), which preferentially binds the mutant form (Kd of 6 μM) compared to wild-type H3.3 .

What tumor-specific patterns of H3F3A and H3F3B mutations have been identified, and how do antibodies facilitate their study?

Tumor-specific patterns of H3F3A and H3F3B mutations exhibit remarkable tissue specificity and clinical importance that can be effectively studied using targeted antibodies:

Mutation Distribution Patterns

This mutually exclusive pattern between tumor types is unprecedented in cancer genetics and provides important diagnostic opportunities .

Methodological Applications of Antibodies

• Diagnostic implementation:

  • Immunohistochemistry with mutation-specific antibodies provides faster and more cost-effective tumor classification compared to sequencing

  • Tissue microarray analysis allows high-throughput screening of tumor cohorts

  • Double-staining protocols can identify cell-specific expression of mutant histones

• Functional characterization:

  • Stable cell line models expressing mutant histones can be validated using specific antibodies

  • Phenotypic effects can be linked to molecular mechanisms (e.g., H3.3K27M promotes glioma cell infiltration through β-catenin/USP1/EZH2 pathway)

  • Comparisons between different mutations provide insights into distinct oncogenic mechanisms

• Experimental validation approaches:

  • Mutant-specific antibodies have been validated using gene sequencing to confirm AAG→ATG codon substitution at K27M

  • Flow cytometry with these antibodies can quantify the percentage of cells carrying mutations

  • Chromatin immunoprecipitation reveals genomic targets affected by mutant histones

• Therapeutic monitoring potential:

  • Antibodies can assess changes in mutant histone expression during treatment

  • Post-treatment tissue analysis can reveal selection pressures on mutant cell populations

  • Liquid biopsy approaches may detect circulating mutant histone proteins

This comprehensive antibody-based approach has fundamentally advanced our understanding of how different histone mutations drive distinct tumor types despite H3F3A and H3F3B encoding identical proteins .

How are H3F3A antibodies advancing understanding of neurodevelopmental disorders?

H3F3A antibodies are becoming increasingly valuable tools in neurodevelopmental research, providing methodological approaches to understand the roles of histone variants in brain development and dysfunction:

• Germline mutation characterization: Recent research has identified that germline mutations in H3F3A and H3F3B cause previously unrecognized neurodevelopmental syndromes. Antibodies specific to these mutant forms allow researchers to study:

  • The expression patterns of mutant histones in different neural cell types

  • The impact on chromatin structure during neurodevelopment

  • Altered interactions with chromatin modifiers and transcription factors

• Developmental expression analysis: Both H3F3A and H3F3B are expressed ubiquitously but show relatively high expression in the brain. Antibody-based approaches enable:

  • Detailed mapping of H3.3 expression in different brain regions during development

  • Comparison between neuronal and glial expression patterns

  • Correlation with critical neurodevelopmental windows

• Epigenetic profiling methodology:

  • ChIP-seq with H3F3A antibodies reveals genomic regions where H3.3 is deposited during neuronal differentiation

  • Sequential ChIP can identify specific modifications on H3.3 in neural progenitors versus mature neurons

  • Integration with transcriptomic data links H3.3 deposition to neurodevelopmental gene expression programs

• Model system validation:

  • Patient-derived iPSCs with H3F3A/B mutations can be differentiated into neural lineages

  • Antibodies confirm appropriate expression of wild-type or mutant H3.3

  • Immunofluorescence reveals alterations in nuclear distribution and chromatin structure

• Therapeutic application potential:

  • Antibodies can monitor changes in histone variant expression following epigenetic therapies

  • High-throughput screening for compounds that normalize aberrant H3.3 deposition patterns

  • Development of diagnostic tools for neurodevelopmental disorders with epigenetic components

The emerging role of H3F3A and H3F3B in neurodevelopment represents an important frontier in epigenetic research, with antibodies providing essential tools for mechanistic studies and potential clinical applications .