HIST1H4A (Ab-31) Antibody

What is HIST1H4A (Ab-31) Antibody and what epitope does it specifically recognize?

HIST1H4A (Ab-31) is a rabbit polyclonal antibody that specifically recognizes the lysine 31 residue of human histone H4. The antibody is generated using a peptide sequence around the site of Lys (31) derived from Human Histone H4 as the immunogen. This specificity makes it valuable for studying post-translational modifications at this particular residue and surrounding regions . The antibody corresponds to the histone H4 protein encoded by the HIST1H4A gene, which has multiple synonyms including H4/a, H4/b, H4/c, among others, reflecting the highly conserved nature of histones across the genome .

What are the validated applications for HIST1H4A (Ab-31) Antibody?

The HIST1H4A (Ab-31) Antibody has been validated for several research applications:

These applications make the antibody versatile for detecting histone H4 in various experimental contexts, from protein expression analysis to chromatin structure studies . The antibody is particularly useful in epigenetic research where precise detection of specific histone modifications is essential.

How should HIST1H4A (Ab-31) Antibody be stored and handled to maintain optimal activity?

For optimal performance, HIST1H4A antibodies should be stored according to manufacturer specifications, typically at -20°C for long-term storage and at 4°C for short-term use. The antibody is generally supplied in liquid format with preservatives such as Proclin 300 (0.03%) to maintain stability . Avoid repeated freeze-thaw cycles as this can compromise antibody performance. When handling, always use sterile technique and avoid contamination. For experiments requiring higher sensitivity, aliquoting the antibody upon receipt is recommended to minimize freeze-thaw cycles and maintain consistent performance across experiments.

How should I determine the optimal antibody concentration for my specific experimental design?

Determining the optimal concentration requires a systematic titration approach:

• Begin with the manufacturer's recommended dilution (typically 1:50-1:200 for immunofluorescence and 1:1000 for Western blot) .

• Perform a dilution series experiment (e.g., 1:50, 1:100, 1:200, 1:500, 1:1000) using positive control samples known to express histone H4.

• Include negative controls lacking the primary antibody to assess background.

• Evaluate signal-to-noise ratio at each concentration.

• Select the dilution that provides the strongest specific signal with minimal background.

For specialized applications like ChIP, optimization may require additional considerations such as chromatin shearing efficiency, antibody binding capacity, and washing stringency. Initial ChIP experiments should include input controls and IgG controls to accurately assess enrichment levels.

What cross-reactivity should I be concerned about when using HIST1H4A (Ab-31) Antibody?

What controls should be included when using HIST1H4A (Ab-31) Antibody in experimental workflows?

A comprehensive control strategy for HIST1H4A antibody experiments should include:

• Positive control: Cell lines or tissues known to express histone H4 (nearly all nucleated cells).

• Negative control: Samples processed identically but without primary antibody application.

• Isotype control: Using rabbit IgG matching the isotype of the primary antibody to assess non-specific binding .

• Peptide competition assay: Pre-incubating the antibody with the immunizing peptide to confirm specificity.

• Knockdown validation: For advanced validation, using histone H4 depletion (though challenging due to its essential nature) or cells with known altered H4 modification states.

For ChIP experiments specifically, include:

• Input chromatin control (typically 5-10% of starting material)

• IgG control to assess non-specific chromatin precipitation

• Positive control loci known to be enriched for histone H4 or the specific modification

How can HIST1H4A (Ab-31) Antibody be effectively used in chromatin immunoprecipitation (ChIP) experiments?

For effective ChIP experiments using HIST1H4A (Ab-31) Antibody:

• Chromatin preparation: Optimize formaldehyde cross-linking time (typically 10-15 minutes) and sonication conditions to generate fragments of 200-500 bp.

• Antibody binding: Use 2-5 μg of HIST1H4A antibody per ChIP reaction with 25-100 μg of chromatin. Incubate overnight at 4°C with rotation to ensure complete binding .

• Washing stringency: Perform high-stringency washes to reduce background while preserving specific interactions. Typically, this involves sequential washes with increasing salt concentration.

• Elution and analysis: After reverse cross-linking and DNA purification, analyze enrichment using qPCR, ChIP-seq, or other appropriate methods.

• Data normalization: Normalize to input controls and compare to IgG controls to accurately quantify enrichment.

For ChIP-seq applications specifically, ensure sufficient sequencing depth (typically 20-30 million reads) and include appropriate bioinformatic analysis to identify regions of enrichment and correlation with other histone marks or genomic features.

How does HIST1H4A (Ab-31) Antibody compare with other histone H4 antibodies in detecting specific post-translational modifications?

HIST1H4A (Ab-31) Antibody specifically targets the lysine 31 position, which distinguishes it from antibodies targeting other modification sites on histone H4:

When designing experiments requiring multiple histone marks, it's important to consider antibody compatibility (host species, isotype) for multiplexing. For co-localization studies using immunofluorescence, select antibodies raised in different host species or use isotype-specific secondary antibodies to avoid cross-reactivity .

What are the advanced applications of HIST1H4A (Ab-31) Antibody in epigenetic research?

The HIST1H4A (Ab-31) Antibody enables several sophisticated epigenetic research applications:

• ChIP-seq profiling: Mapping genome-wide distribution of histone H4 lysine 31 modifications to identify regulatory regions and correlation with gene expression.

• Sequential ChIP (ChIP-reChIP): Identifying genomic regions containing multiple histone modifications by performing sequential immunoprecipitations with different histone mark antibodies.

• ChIP-mass spectrometry: Combining ChIP with mass spectrometry to identify proteins associated with histone H4 and its modifications.

• Single-cell epigenomics: When combined with emerging single-cell technologies, enabling the study of epigenetic heterogeneity within cell populations.

• Dynamics of histone modifications: Using the antibody in time-course experiments to track changes in histone modifications during cellular processes such as differentiation, cell cycle progression, or response to environmental stimuli.

These advanced applications often require optimization beyond standard protocols and may benefit from specialized techniques such as CUT&RUN or CUT&Tag for higher resolution and lower background .

What are common issues encountered when using HIST1H4A (Ab-31) Antibody in Western blotting and how can they be resolved?

Common Western blotting issues and their solutions include:

• Weak or absent signal:

  • Increase antibody concentration (try 1:500 instead of 1:1000)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Ensure proper protein extraction from nuclear fraction

  • Use enhanced chemiluminescence detection systems for higher sensitivity

  • Check transfer efficiency with Ponceau S staining

• High background:

  • Increase blocking time or concentration (5% BSA or milk)

  • Increase washing duration and number of washes

  • Decrease antibody concentration

  • Use freshly prepared buffers

  • Ensure membrane is completely submerged during antibody incubation

• Multiple bands:

  • Histone H4 should appear at approximately 11 kDa

  • Additional bands may represent post-translationally modified forms

  • Higher molecular weight bands could indicate histone aggregates or cross-linked complexes

  • Optimize sample preparation (use histone extraction protocols with acid extraction)

  • Include protease and phosphatase inhibitors in extraction buffers

• Inconsistent results:

  • Standardize protein loading (10-20 μg total protein)

  • Use fresh samples or properly stored frozen aliquots

  • Maintain consistent transfer conditions

  • Document lot-to-lot antibody variation with control samples

How can I validate the specificity of HIST1H4A (Ab-31) Antibody results in my experimental system?

To validate antibody specificity:

• Peptide competition assay: Pre-incubate the antibody with excess immunizing peptide before application to your samples. Specific signals should be significantly reduced or eliminated.

• Correlation with known H4 distribution patterns: Results should be consistent with established patterns of histone H4 localization (nuclear, associated with chromatin).

• Multiple detection methods: Confirm findings using alternative techniques (e.g., verify Western blot results with immunofluorescence).

• Positive and negative controls: Include cell types or tissues with known expression levels of histone H4 or the specific modification being studied.

• Comparison with alternative antibodies: Using a different antibody targeting the same epitope should yield similar results.

• Mass spectrometry validation: For advanced validation, confirm the presence and modification state of the target histone peptide using mass spectrometry analysis.

These validation approaches increase confidence in the specificity of observed signals and help distinguish true biological findings from technical artifacts .

What considerations are important when using HIST1H4A (Ab-31) Antibody in multiplexed immunofluorescence experiments?

For successful multiplexed immunofluorescence:

• Antibody compatibility: When combining multiple primary antibodies, they should be from different host species (e.g., rabbit anti-HIST1H4A with mouse anti-other target) to allow for species-specific secondary antibodies.

• Spectral separation: Choose fluorophores with minimal spectral overlap for secondary antibodies. Standard combinations include FITC/Alexa 488 (green), TRITC/Alexa 594 (red), and DAPI (blue) for nuclear counterstaining.

• Sequential staining: If antibodies are from the same species, consider sequential staining with complete blocking between rounds or use directly conjugated primary antibodies.

• Controls for cross-reactivity: Include single-stained controls to assess potential cross-reactivity between antibodies and non-specific binding of secondary antibodies.

• Fixation optimization: Different targets may require different fixation methods. Histone antibodies typically work well with paraformaldehyde fixation (4%, 10-15 minutes).

• Signal amplification: For low-abundance modifications, consider using signal amplification methods such as tyramide signal amplification (TSA).

• Quantification considerations: For quantitative analysis, include fluorescence intensity calibration standards and control for potential autofluorescence .

How should researchers interpret variations in histone H4 staining patterns across different cell types or treatments?

When interpreting variations in histone H4 staining:

What statistical approaches are recommended for analyzing ChIP data generated using HIST1H4A (Ab-31) Antibody?

For robust statistical analysis of ChIP data:

• Normalization methods:

  • Percent of input: Normalize ChIP signal to input control (typically 5-10% of starting material)

  • Fold enrichment over IgG: Compare specific antibody signal to non-specific IgG control

  • RPKM normalization for sequencing data: Normalize for sequencing depth and region length

• Replication requirements:

  • Minimum of 3 biological replicates recommended

  • Technical replicates (qPCR duplicates/triplicates) to assess measurement precision

• Statistical tests:

  • t-test or ANOVA for comparing enrichment between conditions at specific loci

  • Multiple testing correction (Benjamini-Hochberg) for genome-wide analyses

  • For ChIP-seq: specialized tools like MACS2, DiffBind, or SICER for peak calling and differential binding analysis

• Quality control metrics to report:

  • Coefficient of variation between replicates (<25% preferred)

  • Signal-to-noise ratio (specific enrichment vs. background)

  • For ChIP-seq: library complexity, read alignment rates, fragment size distribution

• Visualization approaches:

  • Genome browser tracks showing raw and normalized signal

  • Heatmaps around features of interest (TSS, enhancers)

  • Aggregate plots showing average profiles around genomic features

How can researchers integrate HIST1H4A (Ab-31) Antibody data with other epigenetic marks to develop comprehensive chromatin state models?

Integrating histone H4 data with other epigenetic marks requires:

• Data integration strategies:

  • Correlation analysis between different histone modifications

  • Chromatin state modeling using tools like ChromHMM or Segway

  • Multi-omics integration with transcriptomic and accessibility data (RNA-seq, ATAC-seq)

• Recommended analysis workflow:

  • Generate consistent ChIP-seq datasets for multiple marks using standardized protocols

  • Process all datasets with the same pipeline to reduce technical variation

  • Perform peak calling and identification of enriched regions

  • Apply unsupervised learning methods to identify recurring patterns of co-occurring marks

  • Annotate identified chromatin states based on genomic features and gene expression data

• Biological validation approaches:

  • Functional validation of predicted regulatory regions using CRISPR-based methods

  • Correlation with gene expression changes in perturbation experiments

  • Comparison with 3D chromatin structure data (Hi-C, ChIA-PET)

• Advanced computational considerations:

  • Account for potential antibody efficiency differences between marks

  • Consider using fixed width bins (e.g., 200bp) across the genome for unbiased state assignment

  • Implement appropriate feature selection and dimensionality reduction methods

  • Validate model robustness through cross-validation approaches

This integrated approach allows researchers to develop comprehensive models of chromatin organization and uncover the functional relationships between different histone modifications, including those detected by HIST1H4A (Ab-31) Antibody.

How is HIST1H4A (Ab-31) Antibody being utilized in single-cell epigenomics research?

Single-cell epigenomics represents a frontier in chromatin research where HIST1H4A antibodies are finding innovative applications:

• Single-cell ChIP technologies:

  • Microfluidic-based single-cell ChIP approaches allow profiling of histone H4 modifications in individual cells

  • Drop-seq compatible ChIP methods enable higher throughput analysis

  • Calibration standards and spike-ins are essential for quantitative comparisons between cells

• CUT&Tag and CUT&RUN adaptations:

  • These antibody-directed transposase or nuclease methods offer higher sensitivity with lower cell numbers

  • Single-cell adaptations allow mapping of histone H4 modifications in individual cells with reduced background

  • Compatible with flow cytometry sorting for selecting specific cell populations before analysis

• Computational challenges and solutions:

  • Sparse data matrices require specialized normalization methods

  • Imputation approaches may be needed to address technical dropouts

  • Trajectory analysis can reveal epigenetic dynamics during cellular transitions

• Multimodal approaches:

  • Combined measurement of histone modifications and transcriptome in the same cell

  • Integration with other single-cell modalities (accessibility, methylation)

  • These approaches require careful antibody validation at the single-cell level

What are the considerations for using HIST1H4A (Ab-31) Antibody in studying the dynamics of histone modifications during cellular processes?

When studying dynamic histone modification changes:

• Temporal resolution considerations:

  • Determine appropriate time points based on the cellular process under study

  • For rapid changes (minutes to hours), consider synchronized cell populations

  • For longer processes (differentiation, development), select representative stages

• Fixation timing and method:

  • Rapid fixation is crucial to capture transient states

  • Standardize fixation protocols for all time points

  • Consider alternative approaches like live-cell imaging with fluorescently tagged readers of histone modifications

• Quantitative analysis approaches:

  • Normalize to appropriate controls at each time point

  • Account for cell cycle effects on histone deposition and modification

  • Use regression analysis or time-series statistical methods to identify significant changes

• Correlation with functional outcomes:

  • Integrate with transcriptomic data at matching time points

  • Connect changes in histone modifications to functional outcomes

  • Consider cause-effect relationships using perturbation experiments

• Reversibility assessment:

  • Design experiments to determine if observed changes are reversible

  • Use inhibitors of writers or erasers to test dependency on continuous enzymatic activity

  • Pulse-chase experiments can reveal turnover rates of specific modifications

How can researchers effectively combine HIST1H4A (Ab-31) Antibody with other emerging technologies for comprehensive epigenetic profiling?

For cutting-edge epigenetic profiling combining HIST1H4A antibody with emerging technologies:

• Proximity ligation approaches:

  • ChIP-PLAC (proximity ligation-assisted ChIP) to study 3D interactions of regions with specific histone marks

  • Combined with Hi-C or HiChIP to relate histone modifications to 3D chromatin structure

  • These methods require optimization of crosslinking conditions for both protein-DNA and protein-protein interactions

• Mass spectrometry integration:

  • ChIP-MS approaches to identify proteins co-occurring with histone H4 modifications

  • Targeted MS to quantify combinations of histone modifications on the same histone tail

  • Requires optimization of sample preparation to maintain PTM integrity

• CRISPR-based epigenome editing:

  • Using dCas9-fused writers or erasers to manipulate H4 modifications at specific loci

  • Validation of antibody specificity by creating controlled epigenetic changes

  • Can help establish causality between histone marks and gene regulation

• Spatial epigenomics:

  • Combining HIST1H4A antibody staining with spatial transcriptomics

  • In situ ChIP approaches for tissue sections

  • Multiplex imaging to map modifications across tissue architecture

• Live-cell epigenetic dynamics:

  • Using antibody-derived recombinant binding domains fused to fluorescent proteins

  • Optogenetic control of histone modifying enzymes combined with antibody-based readouts

  • These approaches require careful validation against fixed-cell antibody staining