HIST1H4A (Ab-1) Antibody

What is HIST1H4A and what role does it play in cellular processes?

HIST1H4A, also known as Histone H4, is one of the core histone proteins that form the protein component of chromosomes in eukaryotic cells. Histones are fundamental proteins rich in arginine and lysine amino acids that have been highly conserved throughout evolution. These proteins function to pack DNA into tight masses of chromatin within the cell nucleus. Two core histones of each class (H2A, H2B, H3, and H4) assemble together with DNA to form nucleosomes, the basic structural units of chromatin .

Histone H4 is particularly important in the regulation of gene expression through post-translational modifications that affect chromatin structure and accessibility. This protein is involved in numerous cellular processes including DNA replication, repair, and transcriptional regulation. Research has shown that Histone H4 can be activated by transcription factors like CCAAT enhancer binding protein β (C/EBPβ) during processes such as mitotic clonal expansion in adipogenesis .

What applications is the HIST1H4A (Ab-1) Antibody validated for?

The HIST1H4A (Ab-1) Antibody has been validated for multiple experimental applications, making it a versatile tool for epigenetic research. According to product specifications, this antibody has been tested and confirmed effective for:

• Chromatin Immunoprecipitation (ChIP)

• Enzyme-Linked Immunosorbent Assay (ELISA)

• Immunofluorescence (IF)

• Immunohistochemistry (IHC)

• Western Blotting (WB)

The antibody has been specifically validated in various experimental systems such as ChIP with HeLa cells treated with Micrococcal Nuclease, Western blot analysis of multiple cell lines (HeLa, HepG2, A549, 293, K562) and tissues (rat spleen, mouse kidney), immunofluorescence staining of HeLa cells, and immunohistochemistry of paraffin-embedded human prostate cancer tissue .

What is the recommended storage protocol for maintaining HIST1H4A (Ab-1) Antibody activity?

Proper storage is critical for maintaining antibody reactivity and experimental reproducibility. For HIST1H4A (Ab-1) Antibody, the recommended storage conditions are:

• 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

For similar antibodies in this class, additional storage recommendations include:

• Centrifuge the product if not completely clear after standing at room temperature

• The antibody is generally stable for several weeks at 4°C as an undiluted liquid

• Dilute only immediately prior to use

• Typical expiration date is one year from the date of opening

What species reactivity has been confirmed for HIST1H4A (Ab-1) Antibody?

HIST1H4A (Ab-1) Antibody has demonstrated reactivity across multiple species, making it suitable for comparative studies. Specifically, the antibody has been confirmed to react with:

• Human

• Mouse

• Rat

This multi-species reactivity is likely due to the high conservation of histone proteins throughout evolution. When designing experiments using this antibody across different species, researchers should consider the high sequence homology of Histone H4 proteins.

What are the recommended dilution ranges for different applications?

Optimal antibody dilution varies significantly depending on the application. For HIST1H4A (Ab-1) Antibody, the recommended dilution ranges are:

• Western Blot (WB): 1:500-5000

• Immunohistochemistry on paraffin-embedded tissues (IHC-P): 1:20-200

• Immunofluorescence (IF): 1:50-200

These ranges should be considered starting points, and researchers should perform titration experiments to determine the optimal concentration for their specific experimental conditions, including sample type, detection method, and instrumentation sensitivity.

How can I validate the specificity of HIST1H4A (Ab-1) Antibody for my experimental system?

Validating antibody specificity is crucial for ensuring experimental reliability. For HIST1H4A (Ab-1) Antibody, consider the following approaches:

• Western blot analysis: The antibody should detect a band at approximately 11.4 kDa (the calculated molecular weight of Histone H4) . Validation has already been performed across multiple cell lines including HeLa, HepG2, A549, 293, and K562, providing a reference for expected results .

• Immunoprecipitation controls: When performing ChIP experiments, include a control using normal rabbit IgG (matching the host species of the antibody) to assess non-specific binding. This control was specifically used in the validation of this antibody's ChIP application with HeLa cells .

• Competition assay: Pre-incubate the antibody with the immunizing peptide (a synthetic peptide containing a sequence from the N-terminal region of histone H4) to confirm that binding is blocked in your detection system .

• Knockout/knockdown validation: Compare antibody staining patterns in wild-type cells versus those with reduced HIST1H4A expression to confirm signal specificity.

What sample preparation methods are optimal for detecting HIST1H4A in different experimental contexts?

Sample preparation varies significantly depending on the experimental application:

For Western blot:

• Whole cell lysates from various cell lines (HeLa, HepG2, A549, 293, K562) have been successfully used with this antibody

• Tissue samples (rat spleen, mouse kidney) can also be effectively analyzed

For ChIP applications:

• Treatment with Micrococcal Nuclease followed by sonication provides optimal chromatin fragmentation

• Immunoprecipitation with 5μg of HIST1H4A (Ab-1) antibody has been validated for effective pulldown

For Immunofluorescence:

• Standard fixation and permeabilization protocols for cultured cells (demonstrated with HeLa cells)

• Use appropriate blocking buffers to minimize background staining

For ELISA applications:

• The antibody has been validated in sandwich ELISA formats

• Detection range of 0.16-10 ng/mL has been established

• Sample types can include tissue homogenates, cell lysates, cell culture supernatants, and other biological fluids

How do quantitative ELISA results for HIST1H4A compare with other detection methods?

The HIST1H4A ELISA system provides quantitative measurement of Histone H4 with high sensitivity and specificity. Key performance metrics include:

• Sensitivity: 0.055 ng/mL

• Detection range: 0.16-10 ng/mL

• Standard curve parameters:

Precision metrics for the ELISA method:

• Intra-assay precision (within an assay): CV% < 8%

• Inter-assay precision (between assays): CV% < 10%

Compared to Western blot or immunofluorescence, ELISA provides more precise quantification but lacks spatial information. ChIP methods, while more complex, provide information about genomic localization that ELISA cannot provide.

What controls should be implemented when performing ChIP experiments with HIST1H4A (Ab-1) Antibody?

For rigorous ChIP experiments using HIST1H4A (Ab-1) Antibody, the following controls are essential:

• Input control: Reserve a portion (typically 5-10%) of the chromatin sample before immunoprecipitation to normalize for differences in starting material.

• Negative antibody control: Use normal rabbit IgG (matching the host species of the primary antibody) to assess non-specific binding. This was specifically used in the validation of this antibody for ChIP applications .

• Positive control region: Include PCR primers for a genomic region known to be associated with histone H4, such as actively transcribed housekeeping genes.

• Negative control region: Include primers for genomic regions where histone H4 is not expected to be enriched, such as gene deserts or repressed genes.

• Technical replicates: Perform at least three independent ChIP experiments to ensure reproducibility, especially when quantifying results using real-time PCR .

What are potential troubleshooting strategies for inconsistent results using HIST1H4A (Ab-1) Antibody?

When encountering inconsistent results with HIST1H4A (Ab-1) Antibody, consider the following troubleshooting approaches:

For Western blot inconsistencies:

• Verify antibody concentration (recommended dilution 1:500-5000)

• Ensure proper sample preparation and loading

• Check transfer efficiency

• Use fresh antibody aliquots to avoid freeze-thaw degradation

• Adjust blocking conditions to reduce background

For Immunohistochemistry/Immunofluorescence issues:

• Optimize fixation and antigen retrieval methods

• Adjust antibody concentration (recommended dilution 1:20-200 for IHC-P, 1:50-200 for IF)

• Extend incubation time or adjust temperature

• Ensure tissues or cells were properly prepared and stored

For ChIP reproducibility problems:

• Verify chromatin shearing efficiency

• Optimize antibody concentration and incubation conditions

• Use fresh aliquots of antibody

• Check for proper controls (IgG control, input controls)

• Ensure consistent cell growth and treatment conditions

For all applications:

• Verify antibody storage conditions (2-8°C short-term, -20°C long-term)

• Check buffer composition (contains 0.03% Proclin 300 and 50% Glycerol)

• Consider lot-to-lot variability and request validation data if available

How can HIST1H4A (Ab-1) Antibody be utilized to investigate histone modifications in gene regulation?

HIST1H4A (Ab-1) Antibody can be leveraged to study histone modifications through several advanced approaches:

• Sequential ChIP (re-ChIP): Use HIST1H4A (Ab-1) Antibody in conjunction with antibodies against specific histone modifications (e.g., acetylation, methylation, phosphorylation) to determine the co-occurrence of modifications on the same histone molecules. This provides insights into the combinatorial histone code.

• ChIP-seq analysis: Combine ChIP using HIST1H4A (Ab-1) Antibody with next-generation sequencing to map the genome-wide distribution of histone H4. The antibody has been validated for ChIP applications in HeLa cells treated with Micrococcal Nuclease , making it suitable for ChIP-seq studies.

• Mass spectrometry: Use HIST1H4A (Ab-1) Antibody to immunoprecipitate histone H4, followed by mass spectrometry analysis to identify and quantify post-translational modifications.

• Time-course experiments: Apply the antibody in temporal studies to track changes in histone H4 occupancy during processes like transcriptional activation. For example, investigating the relationship between C/EBPβ and histone H4 during adipogenesis, as suggested by research showing C/EBPβ activation of histone H4 during mitotic clonal expansion .

• ChIP-qPCR at specific regulatory elements: Use the antibody to analyze histone H4 occupancy at promoters, enhancers, and other regulatory elements in relation to gene expression changes.

What are the methodological considerations for studying C/EBPβ-mediated activation of histone H4?

Research has shown that CCAAT enhancer binding protein β (C/EBPβ) activates histone H4 during mitotic clonal expansion in adipogenesis . When investigating this relationship using HIST1H4A (Ab-1) Antibody, consider these methodological approaches:

• Temporal analysis: Design time-course experiments to capture the dynamics of C/EBPβ binding and subsequent histone H4 activation during adipocyte differentiation.

• ChIP-reChIP: Perform sequential ChIP first with C/EBPβ antibody followed by HIST1H4A (Ab-1) Antibody to identify genomic regions where both proteins co-localize.

• Gene expression correlation: Combine ChIP data using HIST1H4A (Ab-1) Antibody with RNA-seq or qRT-PCR to correlate histone H4 occupancy with changes in gene expression during adipogenesis.

• C/EBPβ binding site analysis: Use bioinformatic approaches to identify C/EBP-binding sites in histone H4 promoter regions, as indicated in research , and validate these sites experimentally using ChIP with HIST1H4A (Ab-1) Antibody.

• Knockdown/knockout studies: Perform C/EBPβ knockdown or knockout experiments and assess changes in histone H4 levels and genomic distribution using HIST1H4A (Ab-1) Antibody in Western blot and ChIP applications.

• Post-translational modification analysis: Investigate how C/EBPβ-mediated activation affects histone H4 modifications by combining HIST1H4A (Ab-1) Antibody with antibodies specific to different histone H4 modifications.

How can HIST1H4A (Ab-1) Antibody be combined with other research tools for comprehensive epigenetic analysis?

For comprehensive epigenetic analysis, HIST1H4A (Ab-1) Antibody can be integrated with multiple complementary approaches:

• Multi-omics integration: Combine ChIP-seq data using HIST1H4A (Ab-1) Antibody with:

  • RNA-seq for correlation with gene expression

  • ATAC-seq for chromatin accessibility analysis

  • DNA methylation data (e.g., bisulfite sequencing)

  • Chromosome conformation capture techniques (Hi-C, 4C, etc.)

• Sequential immunoprecipitation: Use HIST1H4A (Ab-1) Antibody in combination with antibodies against:

  • Other histone variants

  • Specific histone modifications

  • Chromatin remodeling complexes

  • Transcription factors like C/EBPβ

• Advanced microscopy: Combine HIST1H4A (Ab-1) Antibody for immunofluorescence (recommended dilution 1:50-200) with:

  • Super-resolution microscopy techniques

  • Live-cell imaging (if conjugated to appropriate fluorophores)

  • FRAP (Fluorescence Recovery After Photobleaching) for dynamics

  • FISH (Fluorescence In Situ Hybridization) for co-localization with specific genomic loci

• Quantitative proteomics: Use HIST1H4A (Ab-1) Antibody for immunoprecipitation followed by:

  • Mass spectrometry to identify interacting proteins

  • PTM analysis to map modification patterns

  • SILAC or TMT labeling for quantitative comparisons between conditions

What strategies can be employed for studying HIST1H4A in different chromatin states?

To investigate HIST1H4A in various chromatin states, researchers can employ these advanced methodological approaches:

• Chromatin fractionation: Separate chromatin into euchromatin and heterochromatin fractions, then analyze HIST1H4A distribution using the antibody in Western blot applications (recommended dilution 1:500-5000) .

• Salt extraction series: Perform sequential salt extractions to isolate histones associated with different chromatin states, followed by Western blot analysis with HIST1H4A (Ab-1) Antibody.

• ChIP-seq with chromatin state analysis: Perform ChIP-seq using HIST1H4A (Ab-1) Antibody and analyze the distribution across different chromatin states defined by:

  • Histone modification patterns

  • Transcriptional activity

  • Replication timing

  • Nuclear compartmentalization

• Single-cell approaches: Adapt HIST1H4A (Ab-1) Antibody for use in single-cell ChIP or CUT&Tag methods to analyze cell-to-cell variability in histone H4 distribution.

• Developmental or differentiation time courses: Track changes in HIST1H4A localization during cellular differentiation processes, such as adipogenesis where C/EBPβ-mediated histone H4 activation has been observed .

• Drug perturbation studies: Analyze how chromatin-modifying drugs (HDAC inhibitors, HMT inhibitors, etc.) affect HIST1H4A distribution and modification using the antibody in multiple applications.

How can researchers optimize ChIP-seq protocols specifically for HIST1H4A (Ab-1) Antibody?

For optimal ChIP-seq results using HIST1H4A (Ab-1) Antibody, consider these specialized protocol optimizations:

• Chromatin preparation: Use Micrococcal Nuclease treatment followed by sonication, as validated for this antibody in HeLa cells . This approach generates fragments of appropriate size while preserving epitope integrity.

• Antibody concentration: Begin with 5μg of HIST1H4A (Ab-1) Antibody per ChIP reaction as validated in the antibody specifications , but perform titration experiments to determine optimal concentration for your specific cell type.

• Cross-linking conditions: Optimize formaldehyde concentration (typically 1%) and cross-linking time (typically 10 minutes) specifically for histone H4, which may require shorter cross-linking times than larger protein complexes.

• Washing stringency: Adjust salt concentration in wash buffers to achieve the optimal balance between specificity and yield. For histone antibodies, slightly more stringent conditions might be needed due to the high abundance of the target.

• Library preparation: Consider using specialized library preparation methods designed for small DNA fragments, as histone-bound DNA often consists of nucleosome-protected fragments (~150 bp).

• Bioinformatic analysis: Implement analytical approaches that account for the broad distribution patterns often seen with histone proteins, as opposed to the sharp peaks observed with many transcription factors.

• Input normalization: Use appropriate input normalization methods that account for the high abundance and genomic distribution of histone H4.