HIST1H4A (Ab-44) Antibody

What is HIST1H4A (Ab-44) Antibody and what epitope does it target?

HIST1H4A (Ab-44) Antibody is a polyclonal antibody that specifically recognizes the peptide sequence around the lysine 44 residue of human Histone H4 protein. This antibody binds to a core component of the nucleosome, which plays a central role in DNA packaging and chromatin structure. The antibody targets a specific post-translational modification site that is biologically significant in the regulation of chromatin dynamics and gene expression. The antibody recognizes the Histone H4 protein (UniProt ID: P62805) and has been developed using a synthetic peptide immunogen corresponding to the region surrounding Lys-44 of the human Histone H4 sequence.

What are the available applications for HIST1H4A (Ab-44) Antibody?

The HIST1H4A (Ab-44) Antibody has been validated for multiple research applications including:

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative antigen detection

• Immunohistochemistry (IHC): Recommended dilution of 1:10-1:100 for tissue sections

• Western Blotting (WB): Recommended dilution ranges from 1:500-5000

• Immunofluorescence/Immunocytochemistry (IF/ICC): Optimal at 5-20 μg/ml

• Chromatin Immunoprecipitation (ChIP): Validated for chromatin studies

Each application requires specific optimization, and researchers should determine the optimal working concentration for their experimental system. The antibody has demonstrated consistent performance across these applications, making it a versatile tool for histone research.

What species reactivity has been validated for this antibody?

The HIST1H4A (Ab-44) Antibody has been tested and confirmed to react with:

• Human (Homo sapiens): Primary target species with validated reactivity

• Mouse: Cross-reactivity confirmed in tissue samples

• Rat: Validated in various tissue extracts

• Cow: Cross-reactivity noted in some product specifications

The highest specificity has been demonstrated with human samples, where the antibody has been extensively characterized. Cross-reactivity with mouse and rat makes this antibody suitable for comparative studies across these mammalian models. Researchers should still perform preliminary validation when using the antibody in a new species context or with unusual sample types.

What are the optimal storage conditions for maintaining antibody activity?

To maintain optimal HIST1H4A (Ab-44) Antibody activity and prevent degradation, researchers should follow these evidence-based storage guidelines:

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

• Long-term storage: Store at -20°C in small aliquots

• Avoid repeated freeze-thaw cycles, which can significantly degrade antibody performance

• The antibody is typically supplied in a buffer containing 50% glycerol with preservatives (0.03% Proclin 300 in PBS, pH 7.4), which helps maintain stability

• Upon receipt, it is recommended to immediately aliquot the antibody and store appropriately

Researchers have reported maintained activity for up to 12 months from the date of receipt when stored under these conditions. Degradation in antibody performance is often associated with improper storage, particularly multiple freeze-thaw cycles.

How should I optimize Western blot protocols for HIST1H4A (Ab-44) Antibody?

For optimal Western blot results with HIST1H4A (Ab-44) Antibody, follow this methodological approach:

• Sample preparation:

  • Use whole cell lysates (HeLa, HepG2, A549, and 293 cells have been successfully tested)

  • Ensure complete protein denaturation (histones require thorough denaturation)

  • Include protease inhibitors to prevent histone degradation

• Gel electrophoresis:

  • Use 15-18% SDS-PAGE gels for optimal resolution of histone proteins

  • Load 10-20 μg of total protein per lane

• Transfer and blocking:

  • Transfer to PVDF membrane (preferable over nitrocellulose for histone proteins)

  • Block with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Primary antibody incubation:

  • Dilute antibody 1:500-1:5000 in blocking buffer

  • Incubate overnight at 4°C for best results

• Detection:

  • Use appropriate HRP-conjugated secondary antibody

  • Expected molecular weight for Histone H4 is approximately 11 kDa

This protocol has been validated with multiple cell lines including HeLa, HepG2, A549, 293, and K562 whole cell lysates, as well as rat spleen and mouse kidney tissues.

What methods are recommended for optimizing Immunohistochemistry with this antibody?

To achieve optimal immunohistochemical staining with HIST1H4A (Ab-44) Antibody, implement the following protocol:

• Tissue preparation:

  • Use freshly fixed paraffin-embedded tissue sections (4-6 μm thickness)

  • Human prostate cancer tissue has been successfully used for validation

• Antigen retrieval:

  • Heat-induced epitope retrieval using citrate buffer (pH 6.0) is recommended

  • Boil sections for 15-20 minutes followed by cooling at room temperature

• Blocking and antibody incubation:

  • Block with 10% normal goat serum for 30 minutes

  • Dilute primary antibody 1:20-1:200 (start with 1:100 and optimize)

  • Incubate overnight at 4°C in a humidified chamber

• Detection system:

  • Use a polymer-based detection system for enhanced sensitivity

  • Counterstain with hematoxylin for nuclear contrast

  • Expected staining pattern: Nuclear (consistent with histone localization)

Researchers should include appropriate positive controls (human prostate tissue has been validated) and negative controls (primary antibody omission) in each experiment. The recommended dilution range is 1:20-1:200, with the optimal concentration determined empirically based on tissue type and fixation conditions.

How can I use HIST1H4A (Ab-44) Antibody in Chromatin Immunoprecipitation (ChIP) experiments?

For successful Chromatin Immunoprecipitation experiments using HIST1H4A (Ab-44) Antibody, implement this validated protocol:

• Cell preparation:

  • Use approximately 4×10⁶ cells per ChIP reaction (HeLa cells have been validated)

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

  • Quench crosslinking with 125 mM glycine

• Chromatin preparation:

  • Treat lysed cells with Micrococcal Nuclease to fragment chromatin

  • Sonicate to achieve fragments of 200-500 bp (verify fragment size by gel electrophoresis)

• Immunoprecipitation:

  • Use 5 μg of HIST1H4A (Ab-44) antibody per IP reaction

  • Include a control reaction with normal rabbit IgG

  • Incubate overnight at 4°C with rotation

• Washing and elution:

  • Perform stringent washing to remove non-specific binding

  • Elute DNA-protein complexes and reverse crosslinks

• Analysis:

  • Analyze the precipitated DNA by quantitative PCR

  • Target regions where Histone H4 modifications are expected

This protocol has been validated in HeLa cells and can be adapted for other cell types. The antibody has demonstrated sufficient specificity for ChIP applications, with minimal background in IgG control samples.

What post-translational modifications of Histone H4 might affect epitope recognition by this antibody?

The HIST1H4A (Ab-44) Antibody targets the region surrounding lysine 44 on Histone H4. Researchers should consider these potential epitope recognition issues:

• Adjacent post-translational modifications that may affect binding:

  • Acetylation of nearby lysine residues (K31, K59)

  • Methylation of arginine residues (R35, R55)

  • Phosphorylation of serine residues (S47)

• Potential interference mechanisms:

  • Steric hindrance from nearby modifications

  • Conformational changes induced by modifications

  • Protein-protein interactions that mask the epitope

• Experimental considerations:

  • Include appropriate controls when studying histone modifications

  • Consider using this antibody in conjunction with modification-specific antibodies

  • Validate epitope accessibility in your experimental system

Histone H4 undergoes extensive post-translational modifications that form part of the "histone code," regulating DNA accessibility and chromatin structure. These modifications may impact the recognition efficiency of the antibody in certain experimental contexts.

How can I use HIST1H4A (Ab-44) Antibody to investigate nucleosome dynamics in chromatin remodeling studies?

To effectively study nucleosome dynamics using HIST1H4A (Ab-44) Antibody:

• Experimental design approaches:

  • Time-course experiments following induction of chromatin remodeling

  • Comparative analysis across different cell states (quiescent vs. proliferating)

  • Combined ChIP-seq and immunofluorescence microscopy analyses

• Methodological recommendations:

  • Use dual immunofluorescence with HIST1H4A (Ab-44) and chromatin remodeling factors

  • Optimize fixation conditions to preserve nuclear architecture

  • Employ live-cell imaging with fluorescently tagged histones as complementary approach

• Data interpretation guidelines:

  • Changes in Histone H4 distribution patterns may indicate nucleosome repositioning

  • Correlation of Histone H4 occupancy with gene expression data

  • Integration with other histone modification data for comprehensive analysis

• Technical considerations:

  • Use dilution ranges of 1:50-1:200 for immunofluorescence applications

  • Include proper controls for specificity (peptide competition)

  • Consider cell cycle synchronization to reduce variability

This antibody enables visualization of Histone H4 distribution patterns in the nucleus and can be used to track changes in chromatin organization during processes such as differentiation, cell cycle progression, or response to environmental stimuli.

What are common causes of background or non-specific staining when using this antibody?

When encountering background or non-specific staining with HIST1H4A (Ab-44) Antibody, consider these potential causes and solutions:

• Common causes of background:

  • Insufficient blocking: Increase blocking time or concentration of blocking agent

  • Excessive antibody concentration: Perform titration experiments to determine optimal dilution

  • Cross-reactivity with similar epitopes: Use peptide competition assays to confirm specificity

  • Inadequate washing: Increase number and duration of wash steps

• Tissue-specific considerations:

  • Endogenous peroxidase activity: Include peroxidase quenching step for IHC

  • Endogenous biotin: Use biotin-free detection systems if using avidin-biotin methods

  • Autofluorescence: Include appropriate quenching steps for IF applications

• Resolution strategies:

  • Optimize antibody dilution (recommended ranges: WB 1:500-5000, IHC 1:20-200, IF 1:50-200)

  • Increase washing stringency with higher salt concentration

  • Use alternative blocking agents (BSA, fish gelatin, or commercial blockers)

  • Include controls without primary antibody to identify secondary antibody issues

The polyclonal nature of this antibody may contribute to some background in certain applications. Careful optimization of protocols for each experimental system is essential for obtaining clean, specific signals.

How can I validate the specificity of HIST1H4A (Ab-44) Antibody in my experimental system?

To rigorously validate the specificity of HIST1H4A (Ab-44) Antibody in your research:

• Recommended validation approaches:

  • Peptide competition assay: Pre-incubate antibody with immunizing peptide

  • Western blot analysis: Confirm single band at expected molecular weight (~11 kDa)

  • siRNA knockdown: Reduced signal should correlate with reduced target expression

  • Use of multiple antibodies targeting different epitopes of the same protein

• Positive controls to include:

  • HeLa, HepG2, A549, 293, or K562 cell lysates (validated for Western blot)

  • Human prostate cancer tissue (validated for IHC)

  • Rat spleen and mouse kidney tissues (cross-reactivity confirmed)

• Documentation and reporting:

  • Include all validation experiments in publications

  • Report antibody catalog number, lot, and dilution used

  • Document any deviations from manufacturer's recommended protocols

• Advanced validation for specific applications:

  • For ChIP applications: Include IgG control and input samples

  • For IF applications: Include peptide competition and secondary-only controls

These validation approaches ensure experimental reproducibility and reliable interpretation of results across different experimental systems.

What strategies can help resolve contradictory results when using HIST1H4A (Ab-44) Antibody across different experimental platforms?

When faced with contradictory results using HIST1H4A (Ab-44) Antibody across different experimental platforms, implement this systematic troubleshooting approach:

• Technical considerations:

  • Compare fixation methods: Different fixatives may affect epitope accessibility

  • Evaluate buffer compositions: Ionic strength and pH can impact antibody binding

  • Assess protein denaturation conditions: Native vs. denatured states influence epitope exposure

  • Review antibody storage and handling: Degradation may affect performance

• Biological variables:

  • Cell/tissue type differences: Expression levels and modifications vary

  • Cell cycle stage: Histone modifications fluctuate during cell cycle

  • Experimental treatments: May induce changes in histone modifications

• Resolution strategies:

  • Perform side-by-side comparisons using standardized samples

  • Implement orthogonal validation using alternative methods

  • Consider epitope masking in certain contexts

  • Use complementary antibodies targeting different regions of Histone H4

• Analytical approaches:

  • Quantitative comparison across platforms with appropriate normalization

  • Statistical analysis to determine significance of differences

  • Integration of results from multiple antibodies targeting the same protein

This systematic approach helps identify the source of discrepancies and determine whether they represent technical artifacts or biologically meaningful differences in epitope accessibility or modification state.

How can HIST1H4A (Ab-44) Antibody be used to study histone modifications in disease models?

HIST1H4A (Ab-44) Antibody offers valuable applications for investigating histone modifications in disease contexts:

• Cancer research applications:

  • Analyze changes in histone H4 distribution in tumor vs. normal tissues

  • Correlate histone patterns with cancer progression and patient outcomes

  • Investigate the effects of epigenetic drugs on histone H4 distribution

• Neurodegenerative disease studies:

  • Examine histone modifications in models of Alzheimer's, Parkinson's, or other neurodegenerative disorders

  • Combine with neuron-specific markers for cell-type specific analysis

  • Correlate with gene expression changes in affected regions

• Methodological approaches:

  • Tissue microarray analysis using IHC (1:20-200 dilution)

  • ChIP-seq to identify genome-wide binding patterns

  • Co-immunoprecipitation to identify interacting partners

  • Multiplexed immunofluorescence to study co-localization with disease markers

• Experimental design considerations:

  • Include appropriate disease and control samples

  • Consider time-course experiments to track disease progression

  • Integrate with functional assays to determine biological significance

This antibody enables the investigation of epigenetic dysregulation in disease states, providing insights into potential diagnostic biomarkers or therapeutic targets.

What are the considerations for using HIST1H4A (Ab-44) Antibody in conjunction with other histone modification-specific antibodies?

When designing multiplex experiments using HIST1H4A (Ab-44) Antibody together with modification-specific histone antibodies:

• Technical compatibility considerations:

  • Host species compatibility: Select antibodies raised in different species or use directly conjugated antibodies

  • Buffer compatibility: Ensure all antibodies perform optimally in the selected buffer system

  • Incubation conditions: Determine whether sequential or simultaneous incubation is preferable

• Experimental design strategies:

  • Sequential ChIP (Re-ChIP) to detect co-occupancy of modifications

  • Multiplex immunofluorescence to visualize modification patterns

  • Western blot stripping and reprobing for sequential detection

• Antibody selection guidelines:

  • Select antibodies validated for the same application

  • Consider using antibodies recognizing different histone modifications (e.g., H4K12ac, H3K9me3)

  • Include antibodies against chromatin-modifying enzymes for functional studies

• Data analysis approaches:

  • Quantitative co-localization analysis for immunofluorescence

  • Correlation of genomic occupancy profiles from ChIP-seq

  • Western blot band intensity analysis for relative modification levels

This integrated approach provides comprehensive insights into the histone code and its functional significance in various biological contexts.

How might HIST1H4A (Ab-44) Antibody be used in emerging chromatin research techniques?

HIST1H4A (Ab-44) Antibody can be integrated into cutting-edge chromatin research methodologies:

• CUT&RUN and CUT&Tag applications:

  • Protocol adaptation: Use as primary antibody in Cleavage Under Targets and Release Using Nuclease (CUT&RUN) or CUT&Tag protocols

  • Advantage: Higher signal-to-noise ratio than traditional ChIP

  • Recommended starting concentration: 5 μg per reaction (optimize empirically)

• Single-cell epigenetic profiling:

  • Application in single-cell CUT&Tag or scATAC-seq workflows

  • Correlation of histone H4 distribution with chromatin accessibility

  • Integration with single-cell transcriptomics for multi-omic analysis

• Super-resolution microscopy:

  • Use in STORM or PALM imaging for nanoscale localization

  • Recommended dilution: Start at 1:100 and optimize

  • Consider direct fluorophore conjugation for improved resolution

• Live-cell imaging applications:

  • Antibody fragments for live-cell applications

  • Correlation with fluorescently tagged histones

  • Dynamic tracking of histone distribution during cell cycle

These advanced applications extend the utility of HIST1H4A (Ab-44) Antibody beyond conventional techniques, enabling researchers to address complex questions about chromatin organization and dynamics with unprecedented resolution and specificity.