HIST1H4A (Ab-5) Antibody

What is HIST1H4A (Ab-5) Antibody and what epitope does it recognize?

HIST1H4A (Ab-5) Antibody is a rabbit polyclonal antibody that specifically recognizes the lysine 5 (Lys5) site on human Histone H4. The antibody is developed using a peptide sequence around the Lys5 residue derived from Human Histone H4 as the immunogen . This antibody is particularly valuable for studying histone modifications, as Lys5 is a key site for acetylation and other post-translational modifications that regulate chromatin structure and gene expression. The antibody has confirmed reactivity with Human and Mouse samples, with some variants also recognizing Rat samples .

What are the validated applications for HIST1H4A (Ab-5) Antibody?

HIST1H4A (Ab-5) Antibody has been validated for multiple research applications through rigorous testing:

The antibody has been extensively validated on various cell lines including HeLa, HepG2, MCF-7, and NIH/3T3, providing researchers with confidence in its specificity and performance across multiple experimental contexts .

How should HIST1H4A (Ab-5) Antibody be stored to maintain optimal activity?

Proper storage is critical for maintaining antibody functionality. For HIST1H4A (Ab-5) Antibody:

• Short-term storage (up to 2 weeks): Maintain refrigerated at 2-8°C

• Long-term storage: Store at -20°C in small aliquots to prevent freeze-thaw cycles

• The antibody remains stable for up to 12 months from the date of receipt when stored properly

• The liquid formulation contains preservatives (0.03% Proclin 300) and stabilizers (50% Glycerol)

Repeated freeze-thaw cycles should be avoided as they can lead to protein denaturation and loss of antibody activity. Creating multiple small working aliquots upon first thaw is recommended for preserving antibody function throughout your research timeline.

How should I optimize Western blot protocols when using HIST1H4A (Ab-5) Antibody?

When optimizing Western blot protocols with HIST1H4A (Ab-5) Antibody, consider these methodological approaches:

• Sample preparation: Since histones are nuclear proteins, ensure efficient nuclear extraction. For histone H4 detection, acid extraction methods (using 0.2M H₂SO₄ or 0.25M HCl) are often more effective than standard RIPA buffer extractions.

• Gel selection: Histone H4 has a calculated molecular weight of approximately 11.4 kDa . Use high percentage (15-18%) gels or specialized Tris-Tricine systems for optimal resolution of such low molecular weight proteins.

• Transfer conditions: Use PVDF membranes (rather than nitrocellulose) with 0.2μm pore size for better retention of small proteins. Consider semi-dry transfer systems with modified buffers containing 20% methanol.

• Antibody dilution: Begin with 1:1000 dilution in 5% BSA or non-fat milk and adjust based on signal strength . For detecting post-translational modifications, BSA is preferred over milk as blocking agent.

• Controls: Include recombinant Histone H4 as a positive control and samples from H4 knockdown cells as negative controls to confirm specificity.

The validated Western blot images available from suppliers show clear detection of Histone H4 in HeLa, HepG2, MCF-7, and NIH/3T3 cell lysates, confirming the antibody's utility across human and mouse cell lines .

What are the critical factors for successful immunohistochemistry using HIST1H4A (Ab-5) Antibody?

For optimal immunohistochemistry results with HIST1H4A (Ab-5) Antibody:

• Fixation protocol: Use 10% neutral buffered formalin fixation followed by paraffin embedding. Overfixation can mask the Lys5 epitope.

• Antigen retrieval: Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 8.0) is essential. Test both to determine optimal conditions for your specific tissue.

• Permeabilization: Ensure sufficient nuclear permeabilization using 0.1-0.3% Triton X-100 to allow antibody access to nuclear histones.

• Antibody concentration: Start with 1:50 dilution and titrate as needed . For paraffin sections, the recommended range is 1:1-100 .

• Incubation conditions: Overnight incubation at 4°C typically yields better results than shorter incubations at room temperature.

• Detection system: Use polymer-based detection systems rather than avidin-biotin systems to reduce background in histone detection.

• Controls: Include a blocking peptide control to confirm specificity. Validated staining shows nuclear localization in paraffin-embedded human small intestine tissue .

What are the recommended procedures for immunofluorescence applications with this antibody?

For successful immunofluorescence applications using HIST1H4A (Ab-5) Antibody:

• Cell preparation: Culture cells on poly-L-lysine coated coverslips for optimal adherence during processing.

• Fixation: Use 4% paraformaldehyde for 15 minutes at room temperature. Avoid methanol fixation which can extract histones.

• Permeabilization: Use 0.2% Triton X-100 in PBS for 10 minutes to ensure nuclear access.

• Blocking: Block with 5% normal serum (from the species of secondary antibody) with 0.1% BSA in PBS for 1 hour.

• Primary antibody: Apply HIST1H4A (Ab-5) Antibody at 1:50-200 dilution and incubate overnight at 4°C.

• Secondary antibody: Use fluorophore-conjugated anti-rabbit IgG at 1:500 dilution.

• Counterstaining: DAPI (1:1000) for nuclear visualization, as shown in validated images of HeLa cells .

• Mounting: Use anti-fade mounting medium to preserve fluorescence signal.

The validated immunofluorescence images demonstrate nuclear localization of Histone H4 in HeLa cells, consistent with its known biological distribution .

How can I verify the specificity of HIST1H4A (Ab-5) Antibody in my experimental system?

To confirm antibody specificity:

• Peptide competition assay: Pre-incubate the antibody with excess immunizing peptide (containing the Lys5 region) before application to your sample. Signal elimination confirms specificity for the target epitope.

• CRISPR/Cas9 controls: While complete knockout of H4 is lethal, cells with CRISPR-mediated mutations at the Lys5 position can serve as controls.

• Recombinant protein controls: Use purified recombinant histone H4 proteins (wild-type and K5-modified versions) in Western blots as positive controls.

• Multiple application validation: Confirm consistent results across different applications (WB, IF, IHC) to strengthen confidence in specificity.

• Cross-reactivity testing: Test the antibody against samples containing related histone proteins to confirm specificity.

• Mass spectrometry validation: For ultimate confirmation, perform immunoprecipitation followed by mass spectrometry to identify the pulled-down proteins.

The reactivity across human and mouse samples indicates evolutionarily conserved epitope recognition, further supporting specificity claims.

What are common technical challenges when using HIST1H4A (Ab-5) Antibody and how can they be overcome?

Common challenges and their solutions include:

• Weak signal in Western blot:

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

  • Extend primary antibody incubation to overnight at 4°C

  • Use enhanced chemiluminescence (ECL) substrates with higher sensitivity

  • Ensure sufficient histone extraction using acid extraction methods

• High background in IHC/IF:

  • Increase blocking time (2 hours instead of 1 hour)

  • Use 5% BSA instead of normal serum for blocking

  • Reduce antibody concentration (try 1:100 instead of 1:50)

  • Include 0.1% Tween-20 in washing steps

  • Extend washing times between incubations

• Non-specific bands in Western blot:

  • Increase washing stringency with higher salt concentrations

  • Optimize blocking conditions with different blocking agents

  • Consider using gradient gels for better resolution

  • Pre-clear lysates before loading

• Variable results between experiments:

  • Standardize cell/tissue fixation protocols

  • Control for histone post-translational modifications by using HDAC inhibitors

  • Prepare single-use antibody aliquots to avoid freeze-thaw cycles

  • Use internal loading controls specific for nuclear proteins

• Cross-reactivity with other histone variants:

  • Use more stringent washing conditions in immunoprecipitation

  • Consider epitope-tagged histone constructs for validation

  • Compare results with other H4-specific antibodies targeting different epitopes

How can HIST1H4A (Ab-5) Antibody be used effectively in ChIP assays to study histone modifications?

For optimal Chromatin Immunoprecipitation (ChIP) using HIST1H4A (Ab-5) Antibody:

• Crosslinking optimization: Use 1% formaldehyde for 10 minutes at room temperature for standard crosslinking. For studying histone modifications, reduce to 0.75% formaldehyde for 5 minutes to avoid masking the Lys5 epitope.

• Sonication parameters: Optimize sonication conditions to generate chromatin fragments of 200-500bp. For histone studies, shorter fragments (100-300bp) often provide better resolution.

• Antibody amount: Use 2-5μg of HIST1H4A (Ab-5) Antibody per ChIP reaction. Pre-clear chromatin with protein A/G beads before adding antibody to reduce background.

• Incubation conditions: Incubate antibody-chromatin mixture overnight at 4°C with rotation to maximize binding.

• Washing stringency: Use progressively more stringent washing buffers (increasing salt concentration from 150mM to 500mM NaCl) to reduce non-specific binding.

• Elution and reversal: Elute bound chromatin with SDS buffer at 65°C and reverse crosslinks overnight at 65°C.

• Controls: Include:

  • Input chromatin (10% of starting material)

  • IgG negative control

  • Positive control targeting abundant histone mark (e.g., H3K4me3 antibody)

• Analysis methods: Analyze purified DNA by qPCR targeting promoters of housekeeping genes for relative enrichment, or perform ChIP-seq for genome-wide profiling.

This antibody is particularly valuable for studying how Lys5 modifications (acetylation, methylation) correlate with gene expression and chromatin accessibility.

What approaches can be used to study Histone H4 Lys5 modifications using HIST1H4A (Ab-5) Antibody?

To investigate Histone H4 Lys5 modifications:

• Comparison with modification-specific antibodies: Use HIST1H4A (Ab-5) Antibody in parallel with antibodies specific for H4K5ac (acetylation), H4K5me (methylation), or other modifications to assess relative abundance of different modifications at this site.

• Drug treatment studies: Treat cells with HDAC inhibitors (TSA, SAHA) or HAT inhibitors to modulate H4K5 acetylation levels and observe changes in antibody binding patterns.

• Co-immunoprecipitation: Use HIST1H4A (Ab-5) Antibody for immunoprecipitation followed by Western blotting with modification-specific antibodies to quantify relative levels of modifications.

• Sequential ChIP (Re-ChIP): Perform sequential immunoprecipitation with HIST1H4A (Ab-5) followed by modification-specific antibodies to identify genomic regions where specific modifications occur.

• Proximity ligation assays (PLA): Combine HIST1H4A (Ab-5) with antibodies against chromatin-modifying enzymes to detect protein-protein interactions in situ.

• Mass spectrometry analysis: Immunoprecipitate H4 using HIST1H4A (Ab-5) Antibody and analyze by mass spectrometry to identify and quantify all modifications present on the purified histones.

• Cell cycle studies: Combine with cell synchronization protocols to track changes in H4K5 modifications throughout the cell cycle.

• Immunofluorescence co-localization: Perform dual immunofluorescence with HIST1H4A (Ab-5) and antibodies against chromatin states (heterochromatin/euchromatin markers) to correlate H4 distribution with chromatin organization.

How can HIST1H4A (Ab-5) Antibody be used in multiplexed imaging approaches to study chromatin dynamics?

For advanced multiplexed imaging with HIST1H4A (Ab-5) Antibody:

• Sequential immunofluorescence: Use HIST1H4A (Ab-5) Antibody in sequential staining protocols with antibody stripping between rounds to visualize multiple targets in the same sample without spectral overlap limitations.

• Mass cytometry (CyTOF): Conjugate HIST1H4A (Ab-5) Antibody with unique metal isotopes for high-dimensional single-cell analysis of histone modifications in heterogeneous cell populations.

• Multiplex immunofluorescence: Combine HIST1H4A (Ab-5) Antibody with tyramide signal amplification (TSA) systems for simultaneous detection of multiple targets with spectral unmixing.

• Super-resolution microscopy: Optimize sample preparation for STORM, PALM, or STED microscopy to visualize Histone H4 distribution at nanometer resolution, revealing sub-nuclear organization beyond the diffraction limit.

• Live-cell imaging compatibility: While HIST1H4A (Ab-5) is not directly suitable for live-cell imaging, correlative approaches can be developed using fixed-cell staining with this antibody after live imaging with fluorescent histone constructs.

• Expansion microscopy: Combine with tissue expansion techniques to physically enlarge samples after antibody labeling, providing enhanced resolution of chromatin structures.

• Clearing techniques: Optimize antibody penetration in cleared tissue samples (CLARITY, iDISCO) for whole-organ imaging of histone distribution in intact tissues.

• Correlative light and electron microscopy (CLEM): Use gold-conjugated secondary antibodies against HIST1H4A (Ab-5) for correlation between fluorescence and electron microscopy imaging of chromatin ultrastructure.

These advanced imaging approaches enable researchers to correlate Histone H4 dynamics with nuclear architecture, gene expression, and cellular states at unprecedented resolution.

How can HIST1H4A (Ab-5) Antibody be integrated into multi-omics studies of epigenetic regulation?

To integrate HIST1H4A (Ab-5) Antibody into multi-omics approaches:

• ChIP-seq + RNA-seq correlation: Perform ChIP-seq using HIST1H4A (Ab-5) Antibody alongside RNA-seq to correlate histone H4 distribution/modifications with transcriptional activity. This combination reveals how H4 occupancy and modifications influence gene expression programs.

• CUT&RUN alternative: Adapt HIST1H4A (Ab-5) Antibody for CUT&RUN (Cleavage Under Targets and Release Using Nuclease) protocols, which offer higher signal-to-noise ratio than traditional ChIP for mapping histone distributions with lower cell numbers.

• HiChIP applications: Combine chromatin immunoprecipitation with proximity ligation (HiChIP) using HIST1H4A (Ab-5) Antibody to simultaneously map H4 occupancy and chromatin interactions, revealing how H4 contributes to 3D genome organization.

• Single-cell approaches: Optimize HIST1H4A (Ab-5) Antibody for single-cell CUT&Tag or scChIP-seq to reveal cell-to-cell heterogeneity in histone H4 distribution and modifications across populations.

• Integrated analysis workflow: Develop computational pipelines to integrate:

  • ChIP-seq data for H4 (using HIST1H4A (Ab-5))

  • ATAC-seq for chromatin accessibility

  • RNA-seq for gene expression

  • Proteomics data for protein interactions

• Spatial epigenomics: Combine with spatial transcriptomics techniques to correlate H4 distribution with gene expression patterns in tissue contexts, preserving spatial information.

This integrative approach provides a comprehensive understanding of how Histone H4 and its modifications contribute to epigenetic regulation across different scales of biological organization.

What considerations are important when designing experiments to investigate histone H4 dynamics during cell differentiation?

When studying histone H4 dynamics during differentiation using HIST1H4A (Ab-5) Antibody:

• Temporal sampling strategy: Design time course experiments with strategic sampling points that capture key differentiation transitions. Critical timepoints include:

  • Undifferentiated state (day 0)

  • Early commitment phase (12-24 hours)

  • Mid-differentiation (days 2-3)

  • Terminal differentiation (system dependent)

• Normalization approaches: As nuclear architecture changes dramatically during differentiation, careful normalization is essential:

  • Use multiple reference genes for qPCR normalization

  • Consider spike-in standards for ChIP-seq

  • Normalize to total histone H4 content when studying specific modifications

• Microscopy optimization: Adjust imaging parameters throughout differentiation as nuclear size and chromatin compaction change:

  • Standardize exposure settings

  • Use z-stack imaging to capture complete nuclear volume

  • Implement deconvolution for improved resolution

• Cell heterogeneity considerations: Implement strategies to address population heterogeneity:

  • Single-cell approaches (flow cytometry, imaging cytometry)

  • Cell sorting based on differentiation markers

  • Pseudotime analysis for asynchronous differentiation

• Differentiation system validation: Ensure the differentiation system is robust:

  • Monitor established differentiation markers

  • Perform functional assays specific to the terminal cell type

  • Assess reproducibility across biological replicates

• Controls and comparisons:

  • Compare results from directed differentiation with spontaneous differentiation

  • Include differentiation inhibitors as negative controls

  • Evaluate alternative differentiation pathways to identify histone H4 patterns specific to particular lineages

This methodological approach enables robust analysis of how histone H4 distribution and modifications orchestrate gene expression programs during cell fate decisions.