H3F3A (Ab-79) Antibody

What is H3F3A and what does the H3F3A (Ab-79) Antibody recognize?

H3F3A is a gene that encodes the histone variant H3.3, a core component of nucleosomes that plays a central role in transcription regulation, DNA repair, replication, and chromosomal stability. The H3F3A (Ab-79) Antibody specifically recognizes the peptide sequence around the di-methyl-Lys (79) site derived from Human Histone H3.3 . This antibody is a polyclonal antibody raised in rabbits and demonstrates reactivity with human and rat samples . Unlike other H3 antibodies that recognize modifications at K79 such as trimethylation or acetylation, this antibody is specific to the dimethylated form of K79 on the H3.3 variant .

What are the recommended applications for the H3F3A (Ab-79) Antibody?

The H3F3A (Ab-79) Antibody has been validated for several experimental applications:

For Western Blotting applications, researchers should optimize the dilution within the 1:200-1:2000 range depending on their sample type and detection method . When using this antibody for chromatin immunoprecipitation (ChIP), it's particularly valuable for investigating H3.3 deposition at specific genomic loci, especially intronic regions where H3.3 may act as a regulator of gene expression .

How should the H3F3A (Ab-79) Antibody be stored and handled?

For optimal performance and longevity of the antibody:

• Store at -20°C or -80°C upon receipt

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

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

• Working dilutions should be prepared fresh before use and can be stored at 4°C for short periods (1-2 days)

Proper storage and handling are critical as degradation of antibody quality can lead to increased background signal, reduced specificity, or complete loss of immunoreactivity.

How does H3F3A differ from other histone H3 variants and their modifications?

The histone H3 family includes several variants with distinct functions:

H3.3 (encoded by H3F3A) is particularly interesting as it can be incorporated into chromatin independently of DNA replication and is often associated with transcriptionally active regions . Lysine 79 modification on H3.3 (the target of this antibody) differs functionally from modifications at the same position on canonical H3 variants, with research suggesting it plays specific roles in gene activation during cellular processes like cancer progression .

How should I design a ChIP experiment using the H3F3A (Ab-79) Antibody?

When designing a Chromatin Immunoprecipitation (ChIP) experiment with the H3F3A (Ab-79) Antibody:

• Sample preparation:

  • Cross-link protein-DNA complexes with 1% formaldehyde for 10 minutes at room temperature

  • Quench with 125mM glycine for 5 minutes

  • Isolate nuclei and sonicate chromatin to generate fragments of 200-500bp

• Immunoprecipitation:

  • Use 5μg of H3F3A (Ab-79) antibody per ChIP reaction

  • Include appropriate controls (IgG negative control, input control)

  • Incubate overnight at 4°C with rotation

• Washing and elution:

  • Perform stringent washes to remove non-specific binding

  • Elute protein-DNA complexes and reverse cross-links

• Analysis:

  • Analyze by qPCR using primers specific to regions of interest

  • For genome-wide analysis, prepare libraries for ChIP-seq

Based on research findings, focus on intronic regions of genes as H3.3 has been shown to occupy intronic regions in cancer cells where it can modify chromatin status and directly activate transcription . For example, studies have demonstrated that H3.3 is deposited at specific intronic regions of genes like GPR87 in lung cancer cells .

What controls should I include when using this antibody for Western blots?

Proper controls are essential for accurate interpretation of Western blot results:

• Positive control:

  • Cell lines known to express H3F3A (e.g., HeLa cells)

  • Acid-extracted histones from these cells ensure enrichment of histone proteins

• Negative control:

  • Cell lines with H3F3A knockdown

  • Peptide competition assay using the immunizing peptide

• Loading control:

  • Total histone H3 antibody to normalize loading

  • Other housekeeping proteins (though may not be ideal for histone studies)

• Specificity controls:

  • Blots with peptides containing modified and unmodified K79 to verify specificity

  • Testing against other histone modifications (K4, K9, K27, etc.) to confirm absence of cross-reactivity

For Western blotting, the predicted molecular weight of histone H3.3 is approximately 15 kDa . Use SDS-PAGE gels with appropriate resolution for low molecular weight proteins (15-18% acrylamide) for optimal separation.

How can I optimize immunofluorescence staining using the H3F3A (Ab-79) Antibody?

For successful immunofluorescence staining:

• Fixation:

  • Fix cells with 2.5-4% formaldehyde for 30 minutes at room temperature

  • For certain applications, methanol fixation may provide better epitope accessibility

• Permeabilization:

  • Use 0.1-0.5% Triton X-100 in PBS for 5-10 minutes to allow antibody access to nuclear proteins

• Blocking:

  • Block with PBS containing 1% BSA for 30-60 minutes

  • Include 0.1% Tween-20 to reduce background

• Primary antibody:

  • Start with a 1:300 dilution of H3F3A (Ab-79) antibody

  • Incubate overnight at 4°C

• Secondary antibody:

  • Use fluorophore-conjugated anti-rabbit antibody (Alexa 488, 568, or 647)

  • Include DAPI to counterstain nuclei

• Controls:

  • Include a negative control omitting primary antibody

  • Perform peptide competition to confirm specificity

Similar protocols have been successful with other histone H3 antibodies as demonstrated in immunofluorescent analysis of human cells where clear nuclear localization was observed .

How can I use the H3F3A (Ab-79) Antibody to study the role of H3.3 in cancer progression?

Recent research has revealed important roles for H3F3A in cancer progression, particularly in lung cancer:

• Expression analysis:

  • Compare H3F3A expression levels between tumor and normal tissues

  • Correlate expression with clinical parameters (stage, survival)

  • Perform immunohistochemistry on tissue microarrays

• Functional studies:

  • Use ChIP with H3F3A (Ab-79) Antibody followed by sequencing to identify genome-wide binding sites

  • Focus on intronic regions of metastasis-related genes

  • Correlate H3.3 binding with gene expression changes

• Mechanistic investigations:

  • Examine chromatin status at H3.3-bound regions using additional histone modification antibodies

  • Perform sequential ChIP (re-ChIP) to identify co-occurring modifications

  • Investigate interactions with chromatin remodeling complexes

Research has demonstrated that H3F3A overexpression promotes lung cancer cell migration by activating metastasis-related genes . H3.3 was found to globally activate gene expression through occupation of intronic regions, which showed characteristics of regulatory DNA elements . These findings suggest that monitoring H3F3A expression levels could serve as a prognostic marker for early-stage lung cancer .

What are the challenges in distinguishing H3F3A (K79) modifications from similar modifications on canonical H3?

Distinguishing between H3.3 and canonical H3 variants presents several technical challenges:

• Sequence similarity:

  • H3.3 differs from H3.1/H3.2 by only 5/4 amino acids

  • The region around K79 is highly conserved between variants

• Antibody specificity:

  • Validate specificity using dot blot analysis with different histone peptides

  • Perform peptide competition assays to confirm binding specificity

  • Test against various modified and unmodified peptides

• Technical approaches:

  • Mass spectrometry can distinguish variants and modifications

  • Sequential immunoprecipitation with variant-specific then modification-specific antibodies

  • Genetic approaches (H3F3A knockdown/knockout) to validate signals

For dot blot validation, researchers can spot increasing amounts (0.2-100 pmol) of peptides containing the respective histone modifications onto membranes and probe with the antibody at 1/20000 dilution . This approach can effectively demonstrate antibody specificity against various histone modifications.

How can I investigate the relationship between H3F3A deposition and intronic regulation of gene expression?

The discovery that H3.3 occupies intronic regions to regulate gene expression opens new research directions:

• Identify target genes:

  • Perform ChIP-seq with H3F3A (Ab-79) Antibody

  • Analyze intronic enrichment patterns

  • Correlate with gene expression data

• Functional validation:

  • Design reporter constructs with and without identified intronic regions

  • Perform site-directed mutagenesis of putative regulatory elements

  • Use CRISPR-Cas9 to delete intronic regions of interest

• Mechanism exploration:

  • Analyze chromatin accessibility at H3.3-bound intronic regions (ATAC-seq)

  • Investigate recruitment of transcriptional machinery

  • Examine long-range chromatin interactions (3C, Hi-C)

Research has shown that H3.3 is deposited at specific intronic regions of genes like GPR87, where it modifies chromatin status and directly activates transcription . This highlights the importance of intronic regions as regulatory elements in gene expression, particularly in contexts like cancer progression.

How should I interpret ChIP-seq data generated using the H3F3A (Ab-79) Antibody?

Proper interpretation of ChIP-seq data requires careful analysis:

• Quality control metrics:

  • Signal-to-noise ratio: aim for >3

  • Library complexity: assess PCR duplicates

  • Peak distribution: evaluate genomic context

• Peak analysis:

  • Examine distribution across genomic features (promoters, gene bodies, introns)

  • Look for enrichment at intronic regions based on known H3.3 binding patterns

  • Create aggregation plots around transcription start sites and gene bodies

• Comparative analysis:

  • Compare with published H3.3 ChIP-seq datasets

  • Integrate with gene expression data

  • Correlate with other histone modifications

When analyzing data, pay special attention to intronic regions as research has shown H3.3 can bind to specific intronic regions where it modifies chromatin status and activates transcription . For example, studies demonstrated H3.3 deposition at a specific intronic region of GPR87 activated its transcription in lung cancer cells .

What are common pitfalls when using histone variant antibodies and how can I overcome them?

Several challenges are common when working with histone variant antibodies:

• Cross-reactivity issues:

  • Solution: Perform dot blot analysis with modified and unmodified histone peptides

  • Test antibody against arrays of histone modifications

  • Include appropriate controls in each experiment

• Low signal-to-noise ratio:

  • Solution: Optimize antibody concentration

  • Increase washing stringency

  • Use highly specific blocking agents

• Batch-to-batch variability:

  • Solution: Validate each new antibody lot

  • Maintain reference samples for comparison

  • Document lot numbers used for each experiment

• Non-specific binding:

  • Solution: Pre-clear lysates/chromatin

  • Include competitors (e.g., salmon sperm DNA for ChIP)

  • Perform peptide competition assays

• Epitope masking:

  • Solution: Try different fixation methods

  • Test alternative epitope retrieval techniques

  • Consider native ChIP for certain applications

Careful validation through dot blot analysis with various peptides can help confirm specificity, as demonstrated in studies with other histone antibodies where peptides containing 0.2-100 pmol of respective histone modifications were spotted onto membranes for testing .

How can I integrate ChIP-seq data from H3F3A studies with other epigenomic datasets?

Integrative analysis provides deeper insights into H3.3 function:

• Multi-omics integration approaches:

  • Combine H3F3A ChIP-seq with RNA-seq to correlate binding with expression

  • Integrate with ATAC-seq to assess chromatin accessibility at binding sites

  • Include DNA methylation data to examine epigenetic crosstalk

• Analytical tools:

  • Use tools like HOMER, ChIPseeker, or GREAT for genomic annotation

  • Apply integrative platforms like Seqmonk or Galaxy

  • Consider machine learning approaches for pattern recognition

• Visualization strategies:

  • Create browser tracks showing H3F3A binding alongside other datasets

  • Generate heatmaps clustered by binding patterns

  • Produce metaplots centered on features of interest

• Biological interpretation:

  • Focus on cell-type specific patterns

  • Examine developmental trajectories

  • Look for disease-associated signatures

Research demonstrates that integrating H3F3A binding data with expression profiles can reveal regulatory relationships, such as how H3.3 deposition at intronic regions correlates with activation of genes like GPR87 in lung cancer . Such integration helped researchers identify H3F3A and GPR87 expression as prognostic markers for early-stage lung cancer .

What are emerging applications for studying H3F3A in single-cell epigenomics?

Single-cell approaches are revolutionizing our understanding of epigenetic heterogeneity:

• Technical advances:

  • Single-cell ChIP-seq adaptations for H3F3A

  • CUT&Tag approaches for improved sensitivity

  • Combined single-cell transcriptome and epigenome profiling

• Biological applications:

  • Studying H3F3A dynamics during cellular differentiation

  • Investigating heterogeneity within tumor microenvironments

  • Mapping cell-state transitions mediated by H3.3

• Analytical frameworks:

  • Trajectory inference from single-cell epigenomic data

  • Integration with spatial transcriptomics

  • Pseudotime analysis of H3F3A deposition patterns

These approaches would be particularly valuable for studying heterogeneity in cancer progression, as H3F3A has been implicated in cancer cell migration and metastasis-related gene activation . Single-cell approaches could reveal how subpopulations of cells with distinct H3.3 patterns contribute to tumor progression.

How might the study of H3F3A inform therapeutic strategies for cancer treatment?

H3F3A biology suggests several potential therapeutic approaches:

• Target identification:

  • H3F3A-regulated genes as drug targets

  • Proteins that facilitate H3.3 deposition

  • Enzymes that modify H3.3 at K79

• Therapeutic strategies:

  • Small molecule inhibitors of H3.3 chaperones

  • Epigenetic drugs targeting H3.3-associated complexes

  • Antisense oligonucleotides targeting H3F3A

• Biomarker applications:

  • H3F3A expression as prognostic indicator

  • Combined H3F3A/GPR87 expression signature

  • H3.3K79 modification status as predictive biomarker

Research has shown that H3F3A and GPR87 expression levels, either alone or in combination, serve as robust prognostic markers for early-stage lung cancer . This suggests potential for developing treatments involving GPR87 antagonists, highlighting how understanding H3F3A biology can lead to novel therapeutic strategies .

What are the implications of H3F3A intronic regulation for understanding gene expression mechanisms?

The discovery of H3.3's role in intronic regulation represents a paradigm shift:

• Conceptual advances:

  • Introns as active regulatory elements rather than passive spacers

  • Histone variant deposition as a mechanism for intronic regulation

  • Integration of chromatin structure with RNA processing

• Methodological approaches:

  • CRISPR screens targeting intronic H3.3 binding sites

  • Massively parallel reporter assays for intronic elements

  • Long-read sequencing to link chromatin states with splicing patterns

• Broader implications:

  • Potential for widespread intronic regulation across the genome

  • Implications for evolution of gene regulation

  • New layers of complexity in disease-associated gene dysregulation

Research has demonstrated that H3.3 globally activates gene expression through occupation of intronic regions in lung cancer cells, where H3.3 binding regions show characteristics of regulatory DNA elements . This suggests intronic regulation by H3F3A may be a target for developing novel therapeutic strategies in cancer and other diseases .