HIST1H4A (Ab-59) Antibody

What is HIST1H4A (Ab-59) Antibody and what specific epitope does it target?

HIST1H4A (Ab-59) Antibody (product code CSB-PA010429OA59nforHU) is a rabbit polyclonal antibody specifically targeting the region around lysine 59 (Lys59) of human Histone H4. This antibody recognizes the unmodified form of the protein at this specific site. It's generated using a peptide sequence around Lys59 derived from Human Histone H4 as the immunogen and has been affinity purified for specificity . The antibody belongs to the IgG isotype and is provided in liquid form, typically in a buffer containing preservatives like 0.03% Proclin 300 and constituents such as 50% Glycerol in 0.01M PBS at pH 7.4 .

What applications has HIST1H4A (Ab-59) Antibody been validated for?

The HIST1H4A (Ab-59) Antibody has been specifically validated for ELISA and immunohistochemistry (IHC) applications, with recommended dilution ranges of 1:10-1:100 for IHC . Experimental validation through immunohistochemistry has been documented in paraffin-embedded human tissues including cervical cancer and placenta samples, using a Leica Bond™ system with high-pressure antigen retrieval in citrate buffer (pH 6.0) . The detection protocol typically involves blocking with 10% normal goat serum, overnight incubation at 4°C, and visualization using biotinylated secondary antibodies with an HRP-conjugated detection system .

How do HIST1H4A antibodies differ based on their target modifications?

Histone H4 antibodies target various post-translational modifications at different lysine residues, each associated with distinct biological functions:

These different modification-specific antibodies enable researchers to study the complex epigenetic code regulating chromatin structure and gene expression .

What controls should be included when using histone modification antibodies like HIST1H4A (Ab-59) in ChIP experiments?

When designing ChIP experiments with histone modification antibodies, multiple controls are essential:

• Input DNA control: Reserve 5-10% of starting chromatin before immunoprecipitation to normalize for differences in starting material and DNA recovery efficiency .

• Negative control antibody: Include an IgG isotype-matched control antibody from the same species to assess non-specific binding .

• Positive control genomic regions: Include primers for loci known to contain your histone modification of interest .

• Negative control genomic regions: Include primers for loci known to lack your modification .

• Peptide competition controls: Pre-incubate antibody with excess modified peptide that mimics the epitope to confirm specificity .

• Cell treatment controls: Include cells treated with histone deacetylase inhibitors (e.g., sodium butyrate) when studying acetylation marks to generate samples with enriched modifications .

For HIST1H4A (Ab-59) antibody specifically, researchers should consider including controls targeting regions where Lys59 is known to be accessible or inaccessible in the chromatin structure based on previous studies .

How should sample preparation be optimized for immunohistochemistry with HIST1H4A (Ab-59) Antibody?

Based on validated protocols for HIST1H4A (Ab-59) Antibody, optimal sample preparation for IHC should include:

• Fixation and embedding: Standard formalin fixation and paraffin embedding (FFPE) protocols are compatible with this antibody .

• Sectioning: Prepare 4-5 μm thin sections mounted on positively charged slides.

• Antigen retrieval: High-pressure antigen retrieval in citrate buffer (pH 6.0) is critical, as demonstrated in validated tissue samples . This step is essential because formalin fixation can mask epitopes through protein cross-linking.

• Blocking: Block with 10% normal goat serum for 30 minutes at room temperature to reduce non-specific binding .

• Antibody dilution: Prepare antibody in 1% BSA solution at dilutions between 1:10-1:100, with initial testing at 1:20 as demonstrated in validation studies .

• Incubation: Incubate the primary antibody overnight at 4°C for optimal binding .

• Detection system: Use a biotinylated secondary antibody followed by visualization with an HRP-conjugated SP system for optimal results .

• Controls: Include both positive control tissues (cervical cancer, placenta) and negative controls (primary antibody omitted) .

What are the recommended protocols for Western blotting using histone H4 antibodies?

For optimal Western blotting with histone H4 antibodies, follow these methodological steps based on validated protocols:

• Sample preparation:

  • Extract histones using specialized histone extraction kits or acid extraction methods .

  • For total histone analysis, 10-20 μg of acid-extracted histones is typically sufficient .

  • For modification-specific detection, enrichment techniques may be necessary.

• Gel electrophoresis:

  • Use 15-18% SDS-polyacrylamide gels to achieve good separation of low molecular weight histone proteins .

  • Load 10-100 ng for immunoblotting depending on antibody sensitivity .

• Transfer conditions:

  • Transfer to PVDF membranes using semi-dry systems (192 mM glycine, 100 mM Tris, 5% methanol for approximately 70 minutes) .

  • Consider specialized transfer conditions for small proteins (histone H4 is approximately 11-14 kDa) .

• Blocking and antibody incubation:

  • Block membranes with 5% non-fat dry milk or BSA in TBST.

  • Use dilutions of 1:500-1:5000 for most histone H4 antibodies , or 1:1000 for general histone H4 antibodies .

• Controls and treatment conditions:

  • Include positive controls, such as HeLa cells treated with histone deacetylase inhibitors (e.g., sodium butyrate) when studying acetylation marks .

  • Consider running recombinant histones with known modifications as standards .

• Detection considerations:

  • Use high-sensitivity ECL substrates for detecting low-abundance modifications.

  • For histone H4 detection, expect bands at approximately 11-14 kDa .

How can researchers validate the specificity of histone modification antibodies like HIST1H4A (Ab-59)?

Validating antibody specificity is critical for histone modification research. Implement these comprehensive approaches:

• Peptide array analysis: Use the MODified™ Histone Peptide Array containing 384 histone tail peptides with 59 different post-translational modifications in various combinations to determine primary binding specificity and effects of neighboring modifications .

• Dot blot/ELISA with modified peptides: Test antibody against a panel of synthetic peptides containing the modification of interest and related modifications to assess cross-reactivity .

• Peptide competition assays: Pre-incubate antibody with increasing concentrations of the target peptide before application in your assay; specific binding should be blocked in a dose-dependent manner .

• Western blot with recombinant histones: Test against recombinant histones with defined modifications and unmodified controls .

• Immunoprecipitation followed by mass spectrometry: Perform IP followed by mass spectrometry analysis to identify all peptides recognized by the antibody .

• Genetic validation: Use cells from genetic knockout models or CRISPR-edited cells lacking the target histone or the enzyme responsible for the modification .

• Sequential ChIP: Perform sequential ChIP with two different antibodies recognizing the same modification to confirm specificity .

For HIST1H4A (Ab-59) Antibody specifically, validation should include testing against peptides with modifications at or near Lys59 to ensure the antibody recognizes only the intended epitope .

How do neighboring histone modifications affect antibody recognition of target epitopes?

Neighboring modifications can significantly impact antibody recognition through several mechanisms:

• Epitope masking: Modifications adjacent to the target epitope can physically block antibody access. For example, acetylation at H4K8 may affect recognition of H4K12 modifications due to their proximity .

• Conformational changes: Modifications can alter the three-dimensional structure of the histone tail, affecting antibody binding. This is particularly relevant for antibodies like HIST1H4A (Ab-59) that target specific structural conformations .

• Charge effects: Modifications that alter charge (acetylation reduces positive charge; phosphorylation adds negative charge) can affect electrostatic interactions between antibody and epitope .

• Combinatorial effects: The histone code hypothesis suggests that combinations of modifications work together to regulate chromatin function. Research using peptide arrays has shown that:

  • Up to 25% of histone antibodies show significant combinatorial effects where secondary modifications alter binding .

  • Recognition of H4K59 by antibodies can be influenced by modifications at distant sites due to changes in histone folding .

What are the major factors affecting reproducibility in ChIP experiments with histone modification antibodies?

Achieving reproducible results in ChIP experiments requires controlling several critical variables:

• Antibody quality and lot-to-lot variation:

  • Different production lots may show variable specificity and sensitivity .

  • Solution: Validate each new lot against a reference standard and maintain detailed records of antibody performance.

• Chromatin preparation and sonication:

  • Inconsistent chromatin fragmentation leads to variable results .

  • Solution: Optimize sonication conditions to achieve consistent fragment sizes (200-500 bp) and verify by gel electrophoresis.

• Cross-linking efficiency:

  • Under or over-crosslinking affects epitope accessibility and ChIP efficiency .

  • Solution: Standardize fixation time, temperature, and formaldehyde concentration.

• Antibody specificity for modified histones:

  • Cross-reactivity with similar modifications produces misleading results .

  • Solution: Validate antibody specificity using peptide arrays and competition assays.

• Technical variations in immunoprecipitation:

  • Inconsistent washing stringency affects signal-to-noise ratio .

  • Solution: Use automated systems or detailed protocols with timed wash steps.

• PCR amplification bias:

  • GC-rich regions amplify less efficiently .

  • Solution: Use quantitative PCR with validated primers and standard curves.

• Data normalization approaches:

  • Different normalization methods yield variable results .

  • Solution: Consistently apply normalization to input DNA and use spike-in controls.

For optimal reproducibility with HIST1H4A antibodies specifically, researchers should standardize cell culture conditions, since histone modifications are sensitive to cell cycle stage, metabolism, and stress signals .

How should researchers interpret apparent discrepancies between different experimental approaches when studying histone H4 modifications?

When faced with conflicting results across different experimental platforms, consider these systematic interpretation strategies:

• Method-specific limitations:

  • ChIP provides in vivo occupancy but has limited resolution (hundreds of base pairs) .

  • Western blotting shows global modification levels but lacks genomic location information .

  • Immunofluorescence reveals nuclear distribution but not precise genomic targets .

  Solution: Triangulate findings using complementary methods; true biological phenomena should be detectable through multiple approaches.

• Antibody-related factors:

  • Different antibodies targeting the same modification may have distinct specificities or sensitivities .

  • Some antibodies are affected by neighboring modifications while others are not .

  Solution: Validate results using multiple antibodies from different sources targeting the same modification.

• Biological context variations:

  • Histone modifications are dynamic and change with cell cycle, differentiation state, and response to stimuli .

  • Modifications can be locally enriched at specific genomic regions despite low global levels .

  Solution: Carefully control experimental conditions and cell synchronization; compare equivalent cell populations.

• Technical considerations:

  • Fixation conditions affect epitope accessibility differently across methods .

  • Different lysis conditions extract different nuclear fractions .

  Solution: Use standardized protocols with appropriate controls for each technique.

When working specifically with HIST1H4A (Ab-59) Antibody, researchers should be particularly aware of how the accessibility of the Lys59 epitope might differ between native and fixed chromatin, potentially leading to discrepancies between techniques requiring different sample preparation methods .

How do different histone H4 modifications interact functionally in the context of gene regulation?

Histone H4 modifications function as part of an integrated epigenetic code, with complex interdependencies:

• Modification crosstalk:

  • H4K5/K8/K12/K16 acetylation marks often co-occur and collectively contribute to transcriptional activation .

  • H4K20 methylation often antagonizes acetylation marks and is associated with transcriptional repression .

  • Different combinations of modifications create unique binding surfaces for effector proteins .

• Sequential modification patterns:

  • Primary modifications often serve as signals for subsequent modifications.

  • For example, H4K16 acetylation may precede and facilitate other acetylation events on the H4 tail .

  • The temporal order of modifications can determine the functional outcome.

• Spatial distribution patterns:

  • H4K20 methylation is enriched at heterochromatic regions and contributes to chromatin compaction .

  • H4 acetylation marks (K5, K8, K12, K16) are typically enriched at active promoters and enhancers .

  • Different genomic elements (promoters, enhancers, gene bodies) display characteristic modification patterns.

• Context-dependent functions:

  • The same modification can have different functions depending on genomic context.

  • H4K59 may play roles in both structural organization and specific regulatory functions depending on its genomic localization .

Researchers can investigate these interactions using sequential ChIP (re-ChIP) techniques to identify co-occurrence of modifications, or by correlating ChIP-seq datasets for different modifications to map their genomic co-localization patterns .

What emerging technologies are enhancing the study of histone H4 modifications beyond traditional antibody-based approaches?

Several cutting-edge technologies are complementing or replacing traditional antibody-based approaches:

• Mass spectrometry-based approaches:

  • Provides quantitative analysis of histone modifications without antibody bias .

  • Can detect novel or unexpected modifications and modification combinations.

  • Middle-down MS approaches allow analysis of intact histone tails, preserving information about co-occurring modifications.

• Engineered reader domains:

  • Recombinant protein domains designed to recognize specific histone modifications.

  • May offer greater specificity than antibodies for certain modifications.

  • Can be engineered for applications like live-cell imaging of chromatin modifications.

• CUT&RUN and CUT&Tag technologies:

  • Higher signal-to-noise ratio than traditional ChIP .

  • Requires fewer cells and less sequencing depth.

  • Compatible with low input samples and single-cell approaches.

• Single-cell epigenomic technologies:

  • Single-cell ChIP-seq, CUT&Tag, and ATAC-seq provide cell-type-specific information.

  • Reveals heterogeneity in histone modification patterns within populations.

  • Allows correlation of chromatin states with transcriptional output at single-cell resolution.

• CRISPR-based epigenome editing:

  • Targeted deposition or removal of specific histone modifications.

  • Allows direct testing of the functional consequences of specific modifications.

  • Can be used to dissect causal relationships between modifications and gene expression.

Researchers studying histone H4 modifications, including those recognized by HIST1H4A antibodies, are increasingly employing these complementary approaches to overcome the limitations of antibody-based methods and gain deeper insights into the functional consequences of histone modifications .

How can researchers address non-specific binding or high background when using histone H4 antibodies in immunostaining applications?

When encountering high background or non-specific staining with histone H4 antibodies like HIST1H4A (Ab-59), implement these systematic troubleshooting approaches:

• Optimize blocking conditions:

  • Increase blocking time to 1-2 hours.

  • Try different blocking agents: 5% BSA, 5-10% normal serum matched to secondary antibody host, or commercial blockers.

  • For HIST1H4A (Ab-59) specifically, 10% normal goat serum has been validated for effective blocking .

• Antibody dilution optimization:

  • Perform a dilution series spanning the recommended range (1:10-1:100 for HIST1H4A Ab-59) .

  • Consider extending primary antibody incubation time while increasing dilution.

  • Always dilute antibody in blocking solution containing 1% BSA .

• Antigen retrieval refinement:

  • Test different pH buffers for antigen retrieval (citrate pH 6.0 vs. EDTA pH 8.0).

  • Adjust retrieval time and temperature.

  • For HIST1H4A (Ab-59), high-pressure retrieval in citrate buffer (pH 6.0) has been validated .

• Washing protocol optimization:

  • Increase number and duration of washes (e.g., 5 washes of 5 minutes each).

  • Add 0.05-0.1% Tween-20 to wash buffers to reduce non-specific binding.

  • Use gentle agitation during washing steps.

• Secondary antibody considerations:

  • Use highly cross-adsorbed secondary antibodies to minimize species cross-reactivity.

  • Reduce secondary antibody concentration.

  • Consider switching detection systems (HRP vs. fluorescent).

• Tissue-specific controls:

  • Include tissues known to have low expression of the target protein.

  • Use competing peptides to confirm specificity.

  • Include samples from relevant knockout models where available.

• Autofluorescence reduction (for IF applications):

  • Pre-treat samples with sodium borohydride or commercial autofluorescence reducers.

  • Use longer wavelength fluorophores to avoid autofluorescence.

  • Implement spectral unmixing during image acquisition.

For HIST1H4A (Ab-59) Antibody specifically, implementing these optimizations while following the validated protocol parameters has been shown to produce specific nuclear staining with minimal background in human tissue sections .

What quality control measures should be implemented when working with histone modification antibodies in research laboratories?

To ensure reliability and reproducibility when working with histone modification antibodies, implement these quality control measures:

• Initial antibody validation:

  • Perform peptide array testing against multiple modifications to confirm specificity .

  • Document results in a laboratory antibody database.

  • Include positive and negative control peptides containing the target modification and related modifications.

• Lot-to-lot validation:

  • Test each new antibody lot against a reference standard.

  • Maintain a reference sample set for comparison across experiments.

  • Document lot numbers and validation results in experimental records.

• Regular performance testing:

  • Periodically test antibody performance using standard samples.

  • Monitor for changes in signal intensity or specificity over time.

  • Establish acceptance criteria for each application (WB, ChIP, IHC).

• Application-specific controls:

  • For Western blotting: Include modified and unmodified controls, molecular weight markers .

  • For ChIP: Include input, IgG controls, positive and negative loci controls .

  • For IHC/IF: Include positive and negative tissue controls, peptide competition controls .

• Storage and handling protocols:

  • Maintain detailed records of freeze-thaw cycles.

  • Aliquot antibodies to minimize freeze-thaw cycles.

  • Store according to manufacturer recommendations (-20°C or -80°C for most histone antibodies) .

• Documentation standards:

  • Maintain antibody validation files with all QC data.

  • Document catalog numbers, lot numbers, and concentrations in all experiments.

  • Record all experimental conditions that might affect antibody performance.

• Protocol standardization:

  • Develop and follow standard operating procedures for each application.

  • Minimize variations in sample preparation, incubation times, and temperatures.

  • Use automated systems where possible to reduce technical variability.

Implementation of these measures ensures that research using histone modification antibodies, including HIST1H4A antibodies, produces reliable and reproducible results .

What factors affect the stability and shelf-life of histone modification antibodies, and how can researchers maximize antibody longevity?

To maximize the functional lifespan of valuable histone modification antibodies like HIST1H4A (Ab-59), researchers should understand and control these key stability factors:

• Storage temperature conditions:

  • Most histone antibodies should be stored at -20°C or -80°C for long-term stability .

  • Working aliquots can be maintained at 2-8°C for up to 2 weeks .

  • Avoid room temperature storage, even briefly.

• Buffer composition effects:

  • Glycerol content of 30-50% helps prevent freezing damage and stabilizes protein structure .

  • Preservatives like 0.03% Proclin 300 or 0.035% sodium azide prevent microbial growth .

  • pH stability (typically pH 7.4) maintains antibody conformation .

• Freeze-thaw cycle management:

  • Each freeze-thaw cycle can reduce antibody activity by 5-25%.

  • Create multiple small aliquots (10-20 μl) upon receipt of new antibodies.

  • Use screw-cap microcentrifuge tubes to minimize evaporation during storage.

• Antibody concentration factors:

  • Higher concentration antibodies (>1 mg/ml) generally show better stability.

  • Avoid diluting stock antibodies until immediately before use.

  • If dilution is necessary, use fresh buffer with stabilizers.

• Contamination prevention:

  • Use sterile technique when handling antibodies.

  • Never return unused antibody to the original container.

  • Use clean pipette tips and tubes for each antibody.

• Light exposure considerations:

  • Minimize exposure to light, especially for fluorophore-conjugated antibodies.

  • Store in amber tubes or wrap containers in aluminum foil.

  • Avoid extended exposure to UV or bright light during procedures.

• Optimal handling practices:

  • Allow antibodies to warm gradually to room temperature before opening.

  • Centrifuge vials briefly before opening to collect liquid at the bottom.

  • Mix gently by flicking or inverting rather than vortexing.

For HIST1H4A (Ab-59) Antibody specifically, the documented storage recommendations include maintaining refrigeration at 2-8°C for up to 2 weeks, and long-term storage at -20°C or -80°C, while avoiding repeated freeze-thaw cycles . Following these guidelines can extend antibody shelf-life from the typical 12 months to 18-24 months without significant loss of activity.