Di-methyl-H3F3A (K79) Antibody

What is Di-methyl-H3F3A (K79) and why is it important in epigenetic research?

Di-methyl-H3F3A (K79) refers to the dimethylation of lysine 79 on histone H3.3A, a specific post-translational modification (PTM) involved in chromatin regulation. Histone H3.3 is a core component of nucleosomes that wrap and compact DNA into chromatin, limiting DNA accessibility to cellular machineries that require DNA as a template. This modification plays a central role in transcription regulation, DNA repair, DNA replication, and chromosomal stability. Histones regulate DNA accessibility through a complex set of post-translational modifications, collectively termed the "histone code," along with nucleosome remodeling . Studying this specific modification provides insights into gene expression patterns and chromatin states in both normal development and disease conditions.

What applications is Di-methyl-H3F3A (K79) antibody suitable for?

Di-methyl-H3F3A (K79) antibody is compatible with multiple experimental applications that are essential for epigenetic research:

The antibody is primarily used for detecting human (Homo sapiens) samples, though cross-reactivity with other species may occur based on sequence homology .

How does H3F3A (K79) dimethylation differ from other histone modifications?

H3F3A (K79) dimethylation is distinct from other histone modifications in several ways. Unlike many other histone methylation sites that occur on the protruding N-terminal tails, K79 is located within the globular domain of histone H3. This positioning makes it particularly interesting as it may influence nucleosome stability differently than tail modifications. The dimethylation state (K79me2) represents an intermediate level of methylation (between mono- and tri-methylation) and may have specific regulatory functions distinct from K79me1 or K79me3. Additionally, H3K79 methylation is often associated with active transcription, unlike repressive marks such as H3K27me3 . Understanding the specific functions of K79me2 versus other methylation states requires antibodies with high specificity for this particular modification.

How specific are commercially available Di-methyl-H3F3A (K79) antibodies?

Antibody specificity is a critical concern for histone modification research. Recent studies have revealed alarming observations regarding the behavior of histone PTM antibodies, including off-target recognition, influence by neighboring PTMs, and inability to distinguish between modification states (mono-, di-, or tri-methyl lysine) . Specifically for methylated lysine antibodies, research has shown that of 38 di- and tri-methyllysine antibodies screened, 16 cross-reacted with lower states of lysine methylation on the target residue, and one recognized a higher state of lysine methylation .

For Di-methyl-H3F3A (K79) antibodies, validation is essential as these antibodies may potentially cross-react with other methylated lysine residues or be affected by neighboring modifications. Before conducting experiments, researchers should perform validation tests using peptide arrays, dot blots with modified and unmodified peptides, or western blots with appropriate controls (such as cells with K79 methyltransferase knockouts) to ensure specificity.

What methods should I use to validate a Di-methyl-H3F3A (K79) antibody before experiments?

Rigorous validation of Di-methyl-H3F3A (K79) antibodies is essential to ensure experimental reliability. The following methodological approaches are recommended:

• Peptide Array Testing: Screen the antibody against a panel of modified histone peptides to assess cross-reactivity with similar modifications (e.g., K79me1, K79me3) or with the same modification at different positions (e.g., K4me2, K9me2, K27me2).

• Mass Spectrometry Validation: Perform immunoprecipitation followed by mass spectrometry analysis to quantify the degree of target enrichment and specificity .

• Knockout/Knockdown Controls: Test antibody reactivity in cells lacking the enzyme responsible for K79 methylation (similar to SET1 knockout tests for H3K4me3 antibodies) .

• Western Blot Analysis: Compare signal between wild-type samples and those with depleted K79 methylation to confirm specificity.

• Dot Blot Titration: Test antibody against decreasing amounts of modified and unmodified peptides to determine sensitivity and specificity.

How do neighboring histone modifications affect Di-methyl-H3F3A (K79) antibody binding?

Neighboring PTMs can significantly influence antibody recognition of Di-methyl-H3F3A (K79). This phenomenon, known as "epitope occlusion" or "adjacent modification interference," can lead to false negative results when a neighboring modification prevents antibody binding or false positive results when a neighboring modification enhances binding affinity.

For K79me2 antibodies, researchers should consider the following potential interferences:

• Acetylation at nearby lysine residues

• Phosphorylation at adjacent serine or threonine residues

• Other methylation sites in proximity

Research has demonstrated that some H3K27me3 antibodies preferentially bind H3K4me3 peptides, and this off-target recognition is strongly enhanced when H3K4me3 is presented in combination with neighboring acetylation marks . Similar effects could occur with K79me2 antibodies, making it essential to test antibody specificity using peptides with combinations of relevant modifications that might occur naturally in your experimental system.

What is the optimal protocol for ChIP using Di-methyl-H3F3A (K79) antibody?

Chromatin Immunoprecipitation (ChIP) with Di-methyl-H3F3A (K79) antibody requires careful optimization. Here is a methodological approach:

• Cross-linking Considerations:

  • For histone modifications within the globular domain like K79me2, cross-linking conditions are particularly important

  • Start with 1% formaldehyde for 10 minutes at room temperature

  • Test different cross-linking times (5-15 minutes) to optimize signal-to-noise ratio

• Sonication Parameters:

  • Aim for chromatin fragments of 200-500 bp

  • Optimize sonication conditions for your specific cell type

  • Verify fragment size by agarose gel electrophoresis

• Immunoprecipitation:

  • Use 2-5 μg of antibody per ChIP reaction

  • Include appropriate controls (IgG, input, no-antibody)

  • Consider testing both native ChIP and cross-linked ChIP protocols

• Washing Conditions:

  • Use stringent washing to reduce background

  • Include high salt washes to minimize non-specific binding

• Analysis Considerations:

  • Perform qPCR on known targets to validate enrichment

  • For ChIP-seq, include spike-in controls for normalization

Research has shown that the choice to cross-link chromatin in a ChIP can significantly impact the resultant immunoprecipitation, especially for modifications like H3K79me2 . In some cases, H3K79me2 antibodies may perform differently under native versus cross-linking conditions, with some antibodies showing modest enrichment under native conditions but failing under cross-linking conditions .

How should I optimize Western blot protocols for Di-methyl-H3F3A (K79) detection?

For optimal Western blot detection of Di-methyl-H3F3A (K79), follow these methodological guidelines:

• Sample Preparation:

  • Extract histones using acid extraction (0.2N HCl or 0.4N H2SO4)

  • Alternatively, use specialized histone extraction kits

  • Include appropriate controls (unmodified H3, total H3)

• Gel Electrophoresis:

  • Use 15-18% SDS-PAGE gels for optimal histone separation

  • Load 5-10 μg of acid-extracted histones or 20-30 μg of whole cell lysate

• Transfer Conditions:

  • Use PVDF membranes (preferred over nitrocellulose for histone proteins)

  • Optimize transfer time and voltage (typically lower voltage for longer time)

• Blocking and Antibody Incubation:

  • Block with 5% BSA (not milk, which contains casein kinases that may interfere)

  • Incubate with primary antibody at 1:1000 to 1:2000 dilution

  • Wash thoroughly to reduce background

• Detection:

  • Use highly sensitive detection systems (ECL-plus or fluorescent secondary antibodies)

  • Perform parallel blots with antibodies against total histone H3 for normalization

When analyzing Western blot results, remember that the expected molecular weight for histone H3 is approximately 17 kDa . To verify specificity, consider including samples with known K79 methylation status or performing peptide competition assays.

What controls should I include in experiments using Di-methyl-H3F3A (K79) antibody?

Including appropriate controls is crucial for experiments using Di-methyl-H3F3A (K79) antibody:

For definitive validation, consider genetic approaches where possible, such as using cells with CRISPR-mediated knockout or knockdown of the enzyme responsible for K79 dimethylation. This approach is supported by research demonstrating loss of signal in cells lacking the relevant histone methyltransferase, as shown with H3K4me3 antibodies in SET1 knockout systems .

How can I distinguish between true H3K79me2 signal and antibody cross-reactivity?

Distinguishing between true H3K79me2 signal and antibody cross-reactivity requires multiple validation approaches:

• Peptide Array Verification: Test your antibody against a comprehensive histone peptide array to identify potential cross-reactive epitopes. Research has shown that some methyllysine antibodies can recognize the same modification at different positions (e.g., H3K9me3 antibodies recognizing H3K27me3) .

• Multiple Antibody Concordance: Use two or more antibodies against H3K79me2 from different vendors or clones. Signals detected by multiple independent antibodies are more likely to represent true H3K79me2.

• Correlation with Enzyme Activity: Modulate the activity of the methyltransferase responsible for K79 dimethylation and observe corresponding changes in antibody signal.

• Mass Spectrometry Validation: Perform mass spectrometry analysis of immunoprecipitated samples to confirm enrichment of the target modification. Research has demonstrated that MS can quantify the degree of target enrichment and specificity of histone antibodies .

• Sequential ChIP: For ChIP experiments, perform sequential ChIP with different antibodies (e.g., first with anti-H3, then with H3K79me2) to increase specificity.

Remember that even well-characterized commercial antibodies may have undocumented cross-reactivity. Research has shown that H3K27me3 antibodies can cross-react with H3K4me3 in budding yeast, which lacks H3K27 methylation , highlighting the importance of rigorous validation.

What are common issues when using Di-methyl-H3F3A (K79) antibody in ChIP experiments?

Common issues with Di-methyl-H3F3A (K79) antibody in ChIP experiments include:

• Low Enrichment: K79me2 is located in the globular domain of H3, which may be less accessible in cross-linked chromatin. Consider:

  • Testing native ChIP conditions without formaldehyde

  • Optimizing sonication to improve epitope exposure

  • Increasing antibody amount or incubation time

• High Background: Non-specific binding can obscure true signals. Address by:

  • Increasing washing stringency with higher salt concentrations

  • Pre-clearing chromatin with protein A/G beads

  • Using more specific antibody clones or batches

• Cross-reactivity: Antibodies may recognize similar epitopes. Mitigate by:

  • Performing peptide competition assays

  • Using knockout controls when available

  • Validating with independent techniques

• Variability Between Antibody Lots: Different lots may have different specificities. Control by:

  • Testing new antibody lots against old ones

  • Maintaining reference samples across experiments

  • Using antibody characterization platforms

• Fixation Effects: Research has shown that some H3K79me2 antibodies perform poorly under cross-linking conditions . If this occurs:

  • Test both native and cross-linked ChIP protocols

  • Adjust cross-linking time and formaldehyde concentration

  • Consider enzymatic fragmentation rather than sonication

To validate ChIP signals, compare enrichment at known positive regions (genes with expected K79me2) versus negative regions, and include appropriate controls in each experiment.

How should I interpret contradictory results between different experimental approaches using Di-methyl-H3F3A (K79) antibody?

When faced with contradictory results between different experimental approaches using Di-methyl-H3F3A (K79) antibody, consider the following methodological analysis strategy:

• Evaluate Antibody Performance in Each Assay:

  • Some antibodies perform well in Western blot but poorly in ChIP and vice versa

  • Research has shown that H3K79me2 antibodies may enrich for targets under native conditions but not under cross-linking conditions

  • Check if epitope accessibility differs between applications

• Consider Technical Differences:

  • Protein denaturation state (native vs. denatured)

  • Epitope accessibility (surface exposure in different techniques)

  • Fixation effects on epitope structure

  • Buffer conditions affecting antibody-epitope interaction

• Biological Context:

  • Cell type-specific differences in histone modification patterns

  • Cell cycle stage affecting global histone modification levels

  • Environmental conditions impacting epigenetic states

• Resolution Approach:

  • Employ orthogonal methods like mass spectrometry to resolve discrepancies

  • Test with multiple antibodies targeting the same modification

  • Validate with genetic approaches (enzyme knockouts)

  • Consider using semi-synthetic nucleosomes with defined modifications for control experiments

• Methodological Reconciliation:

  • Standardize extraction methods across experiments

  • Normalize data appropriately for each technique

  • Establish clear positive and negative controls for each method

When reporting contradictory results, present data from all methods with appropriate controls and discuss potential technical or biological explanations for the discrepancies.

How can Di-methyl-H3F3A (K79) antibody be used in multiplexed histone modification analysis?

Multiplexed analysis of histone modifications including Di-methyl-H3F3A (K79) enables researchers to understand the relationships between different epigenetic marks. Here are methodological approaches for multiplexed analysis:

• Sequential ChIP (Re-ChIP):

  • Perform initial ChIP with one antibody (e.g., H3K79me2)

  • Elute the protein-DNA complexes

  • Perform a second round of ChIP with another antibody

  • This identifies genomic regions containing both modifications

• Mass Spectrometry-Based Approaches:

  • Use antibody enrichment followed by MS analysis

  • Quantify co-occurrence of modifications on the same histone molecules

  • Research has demonstrated that MS can quantify enrichment of histone modifications using ChIP-grade antibodies

• ChIP-seq with Parallel or Sequential Immunoprecipitations:

  • Perform parallel ChIP-seq experiments with different histone mark antibodies

  • Compare genomic distributions to identify overlapping or mutually exclusive patterns

  • Use comprehensive bioinformatic analysis to correlate modification patterns

• CUT&RUN or CUT&Tag with Antibody Panels:

  • These newer techniques offer higher signal-to-noise ratios

  • Can be performed with smaller cell numbers

  • Allow for multiplexed analysis with appropriate controls

• Imaging-Based Approaches:

  • Immunofluorescence with multiple antibodies

  • Proximity ligation assays to detect co-occurrence of marks

  • Super-resolution microscopy for detailed nuclear distribution

What are the considerations for using Di-methyl-H3F3A (K79) antibody in single-cell epigenomic analyses?

Single-cell epigenomic analyses with Di-methyl-H3F3A (K79) antibody present unique challenges and opportunities:

• Antibody Sensitivity Requirements:

  • Single-cell techniques require highly sensitive antibodies

  • Signal amplification methods may be necessary

  • Batch testing of antibodies is crucial for consistent results

• Protocol Adaptations:

  • Miniaturization of ChIP protocols (micro-ChIP or nano-ChIP)

  • Integration with microfluidic platforms

  • Modified fixation to maintain cellular integrity while enabling antibody access

• Technical Challenges:

  • Limited material from single cells requires highly specific antibodies

  • Background signal becomes more problematic at the single-cell level

  • Need for specialized controls to account for technical variation

• Data Analysis Considerations:

  • Higher data sparsity compared to bulk methods

  • Need for specialized computational approaches

  • Integration with other single-cell data types (transcriptomics, etc.)

• Validation Strategies:

  • Correlate with bulk population data

  • Use spike-in controls for normalization

  • Perform parallel analyses with orthogonal methods

For single-cell applications, antibody specificity is even more critical than in bulk assays. Consider using antibodies validated specifically for CUT&Tag or other single-cell compatible techniques, and always include appropriate controls to distinguish true signal from technical artifacts.

How do different cell fixation methods affect Di-methyl-H3F3A (K79) antibody binding in chromatin studies?

Cell fixation methods significantly impact Di-methyl-H3F3A (K79) antibody binding in chromatin studies due to the location of K79 within the globular domain of histone H3. Consider these methodological factors:

• Formaldehyde Cross-linking:

  • Standard 1% formaldehyde may over-fix and mask the K79me2 epitope

  • Research has shown that some H3K79me2 antibodies perform poorly under cross-linking conditions

  • Titrate formaldehyde concentration (0.1-1%) and fixation time (5-15 minutes)

• Native Conditions (No Cross-linking):

  • May preserve antibody epitopes better for some modifications

  • Studies have shown that some H3K79me2 antibodies enrich targets under native conditions but not cross-linked conditions

  • Consider testing both approaches in parallel

• Alternative Fixatives:

  • Disuccinimidyl glutarate (DSG) in combination with formaldehyde

  • Ethylene glycol bis(succinimidyl succinate) (EGS)

  • Methanol or ethanol fixation for certain applications

• Fixation Effects on Chromatin Structure:

  • Over-fixation can compact chromatin and reduce accessibility

  • Under-fixation may not preserve protein-DNA interactions

  • Different cell types may require optimized fixation protocols

• Optimization Strategy:

  • Test a matrix of conditions (fixative type, concentration, time)

  • Evaluate epitope accessibility via dot blot or Western blot

  • Perform ChIP-qPCR on known targets to compare efficiency