HIST1H4A (Ab-35) Antibody

Basic Research Applications

• What is HIST1H4A and what does the (Ab-35) designation indicate?

HIST1H4A is one of several genes encoding histone H4, a critical core histone protein that forms part of the nucleosome structure. Histone H4 is highly conserved across species and plays essential roles in chromatin organization and gene regulation. The HIST1H4A gene product is functionally identical to other H4 variants (including HIST1H4B through HIST1H4L) .

The "(Ab-35)" designation refers to the specific epitope this antibody recognizes - a peptide sequence surrounding arginine at position 35 in the human histone H4 protein . This epitope location is within the globular domain rather than the N-terminal tail where most post-translational modifications occur, making it potentially useful for detecting total H4 regardless of modification state.

• What validated applications can the HIST1H4A (Ab-35) Antibody be used for?

The HIST1H4A (Ab-35) Antibody has been validated for multiple research applications including:

Each application utilizes different aspects of the antibody's binding characteristics, with ChIP being particularly valuable for studying histone-DNA interactions and chromatin structure .

• How do I validate the specificity of HIST1H4A (Ab-35) Antibody in my experimental system?

Rigorous validation is essential for obtaining reliable results with any antibody. For HIST1H4A (Ab-35) Antibody, implement these key validation strategies:

• Western blot analysis: Verify a single band at approximately 11 kDa corresponding to histone H4

• Peptide competition assay: Pre-incubate the antibody with excess immunizing peptide (Arg35 region of H4); signal elimination confirms specificity

• Knockout/knockdown controls: Compare signal between wild-type cells and those with reduced H4 expression

• Cross-reactivity assessment: Test against other histones (H2A, H2B, H3) to confirm lack of cross-reactivity

• Immunofluorescence colocalization: Verify nuclear localization and pattern consistency with other H4 antibodies

These validation steps ensure that experimental observations can be confidently attributed to histone H4 detection rather than non-specific binding or artifacts.

Experimental Design and Optimization

• How should I optimize ChIP protocols when using HIST1H4A (Ab-35) Antibody?

Optimizing ChIP protocols with HIST1H4A (Ab-35) Antibody requires attention to several key parameters:

Cross-linking Optimization:

• For histone H4 studies, standard formaldehyde cross-linking (1% for 10 minutes) is typically sufficient

• Over-fixation can mask epitopes and reduce antibody binding efficiency

Sonication Parameters:

• Aim for chromatin fragments between 200-500 bp for optimal resolution

• Verify fragmentation efficiency via gel electrophoresis before proceeding

Antibody Considerations:

• Use 2-5 μg of antibody per ChIP reaction

• Include IgG control to assess non-specific binding

• Consider using H4-specific genomic regions (like HIST1H4E promoter) as positive controls

Washing Stringency:

• Balance between reducing background (more stringent washes) and maintaining specific signal

• Include matched IgG control to assess non-specific binding

Based on research using H4-targeting antibodies, careful optimization can yield valuable insights into histone H4 distribution patterns and their relationship with transcriptional regulation mechanisms .

• What approaches can be used to study H4 tail interactions with chromatin remodelers using this antibody?

The histone H4 tail plays a critical role in chromatin dynamics through interactions with various remodeling complexes. Research has shown that "ACF senses linker DNA length through an interplay between its accessory and catalytic subunits mediated by the histone H4 tail of the nucleosome" . The HIST1H4A (Ab-35) Antibody can be integrated into several approaches to study these interactions:

Co-Immunoprecipitation (Co-IP):

• Use the antibody to pull down H4 and associated remodeling complexes

• Western blot or mass spectrometry can identify interacting partners

• Note: Since the epitope is at Arg35, interactions occurring specifically at the N-terminal tail might not be disrupted

Proximity Ligation Assay (PLA):

• Combine HIST1H4A (Ab-35) Antibody with antibodies against chromatin remodelers

• PLA signal indicates close physical proximity (<40 nm) between the proteins

ChIP-reChIP:

• Perform sequential immunoprecipitation with HIST1H4A (Ab-35) Antibody followed by antibodies against remodeling complex components

• This identifies genomic regions where both H4 and specific remodelers co-localize

Research has demonstrated that for nucleosomes with short linker DNA, proteins like Acf1 preferentially bind to the H4 tail, while "as the linker DNA lengthens, Acf1 shifts its binding preference to the linker DNA, freeing the H4 tail" . These dynamic interactions can be further explored using the HIST1H4A (Ab-35) Antibody in carefully designed experiments.

• How does the epitope recognized by HIST1H4A (Ab-35) Antibody relate to HAT1-dependent acetylation sites?

The epitope recognized by HIST1H4A (Ab-35) Antibody—centered around Arginine 35—has an important spatial and functional relationship to HAT1-dependent acetylation sites on histone H4:

Spatial Relationship:

• HAT1 primarily acetylates newly synthesized histone H4 at lysines 5 and 12 (H4K5Ac and H4K12Ac)

• The Arg35 epitope is located in the globular domain of H4, distinct from these N-terminal tail acetylation sites

• This positioning makes the antibody valuable for detecting total H4 regardless of acetylation status

Functional Implications:

• Research has shown that "HAT1 depletion led to substantial depletion of newly synthesized histone H3 and H4 levels," suggesting a coordination mechanism between H4 acetylation and production

• The antibody can be used to monitor total H4 levels when investigating HAT1's role in histone dynamics

Experimental Applications:

• For studying HAT1-mediated acetylation, use HIST1H4A (Ab-35) Antibody alongside modification-specific antibodies (H4K5Ac, H4K12Ac)

• In ChIP experiments, compare HIST1H4A (Ab-35) signal with HAT1-dependent acetylation marks to distinguish between changes in H4 occupancy versus modification levels

According to studies, "HAT1-dependent H4K12Ac and H4K5Ac marks identified 2590 ± 3163 and 14012 ± 9599 peaks, respectively" in genome-wide analyses , highlighting the importance of differentiating between H4 occupancy and specific modifications when interpreting experimental results.

Advanced Research Applications

• How can HIST1H4A (Ab-35) Antibody be used to study histone modifications in cancer research?

The HIST1H4A (Ab-35) Antibody can be strategically employed in cancer research to investigate histone H4 dynamics and their relationship to oncogenic processes:

Total H4 as Normalization Control:

• When studying specific H4 modifications (e.g., H4K5Ac, H4K12Ac), this antibody serves as a control for total H4 levels

• This normalization is crucial when comparing modification levels between normal and cancer tissues

ChIP-seq Analysis of H4 Occupancy:

• Map genome-wide H4 distribution in cancer cells compared to normal counterparts

• Identify regions with altered nucleosome positioning that may affect gene expression

• Correlate H4 distribution with cancer-specific transcriptional programs

Integration with HAT1 Studies:

• HAT1 coordinates histone production and acetylation via H4, with implications for cell proliferation

• Use the antibody to study how HAT1 depletion affects total H4 levels in cancer cell lines

• Investigate the regulatory relationship between histone H4 and cancer-associated transcription factors like HNF4A in liver cancer

Histone Variant Expression:

Research in liver cancer has demonstrated that histone modification patterns correlate with specific cancer subtypes and patient outcomes . The HIST1H4A (Ab-35) Antibody can be employed to investigate the relationship between total H4 levels, specific modifications, and cancer progression.

• What are the challenges in interpreting ChIP-seq data generated using HIST1H4A (Ab-35) Antibody?

Interpreting ChIP-seq data from experiments using HIST1H4A (Ab-35) Antibody presents several unique challenges that researchers should address:

Distinguishing Signal Origins:

• Since the antibody recognizes multiple H4 variants (HIST1H4A through HIST1H4L), peaks represent aggregate H4 occupancy rather than variant-specific distributions

• Complementary approaches may be needed to resolve variant contributions

Disentangling Occupancy from Modification:

• H4 is subject to numerous post-translational modifications that may affect antibody binding efficiency

• Regions with heavy modifications might show different signal intensities even with similar H4 levels

• Research has shown that acetylation patterns at H4K5 and H4K12 have regulatory roles in transcription

Normalization Considerations:

• Unlike transcription factor ChIP-seq, histone H4 is ubiquitous across the genome

• Standard normalization methods may not account for global changes in H4 levels between conditions

• Consider using spike-in controls for accurate between-sample comparisons

Data Interpretation Framework:

• H4 peaks should be interpreted in the context of chromatin accessibility and gene expression data

• Integrative analysis with other histone marks provides more comprehensive insights

• Look for correlation with known H4-dependent processes such as HAT1-regulated gene expression

A study investigating HAT1 function performed "ChIP-seq for the HAT1-dependent H4K12Ac and H4K5Ac marks" and identified thousands of peaks . When analyzing H4 ChIP-seq data, similar sophisticated approaches to peak calling and differential binding analysis should be employed, with careful attention to appropriate controls and normalization methods.

Data Analysis and Troubleshooting

• How do I troubleshoot inconsistent results when using HIST1H4A (Ab-35) Antibody across different experiments?

When encountering inconsistent results with HIST1H4A (Ab-35) Antibody across experiments, implement this systematic troubleshooting approach:

Sample Preparation Variables:

• Fixation conditions: Duration and temperature of fixation can affect epitope accessibility

• Extraction method: For histones, extraction protocols significantly impact yield and quality

• Storage conditions: Repeated freeze-thaw cycles of antibody or samples can reduce activity

Antibody-Specific Factors:

• Lot-to-lot variation: Compare lot numbers and request technical information from manufacturer

• Antibody age/storage: Activity may decrease over time, especially with improper storage

• Working concentration: Re-titrate antibody for each new experimental setup or application

Technical Execution:

• Protocol consistency: Document detailed protocols and minimize variation between experiments

• Equipment calibration: Ensure consistent performance of critical equipment (e.g., sonicators for ChIP)

• Reagent quality: Prepare fresh buffers and verify pH, which is critical for antibody-epitope interactions

Biological Variability:

• Cell cycle effects: Histone dynamics vary throughout the cell cycle; synchronize cells when possible

• Cell density/confluence: Standardize culture conditions to minimize epigenetic variation

• Passage number: Limit cell passage variation between experiments

By methodically addressing these variables and performing appropriate controls (including IgG controls and known positive samples), researchers can identify sources of inconsistency and establish more reliable experimental conditions for HIST1H4A (Ab-35) Antibody applications.

• What statistical approaches are recommended for analyzing quantitative data from HIST1H4A (Ab-35) Antibody experiments?

Analyzing quantitative data from experiments using HIST1H4A (Ab-35) Antibody requires appropriate statistical approaches tailored to the specific application:

For Western Blot Densitometry:

• Normalize H4 signal to loading controls (β-actin, GAPDH) or total protein stains

• Apply log transformation for data that spans multiple orders of magnitude

• Use paired t-tests for before/after comparisons or ANOVA for multiple conditions

• Report fold-change with 95% confidence intervals rather than just p-values

For ChIP-qPCR Analysis:

• Calculate percent input or fold enrichment over IgG control

• Apply non-parametric tests (Mann-Whitney U) if normality cannot be assumed

• Use multiple reference regions for normalization to account for technical variation

• Include biological replicates (minimum n=3) for robust statistical analysis

For ChIP-seq Data:

• Employ specialized software packages designed for histone ChIP-seq analysis

• Account for global differences in histone levels when normalizing between conditions

• Use false discovery rate (FDR) correction for multiple testing

• Consider biological replicates essential for reliable differential binding analysis, as demonstrated in studies of HAT1-dependent H4 acetylation marks

For Immunofluorescence Quantification:

• Measure integrated intensity rather than maximum intensity for more accurate quantification

• Apply background subtraction based on negative control samples

• Use mixed-effects models to account for cell-to-cell variability within and between samples

• Consider nuclear area normalization when comparing cells of different sizes