HIST1H4A (Ab-79) Antibody

What is HIST1H4A (Ab-79) Antibody and what are its key specifications?

The HIST1H4A (Ab-79) Polyclonal Antibody is a rabbit-hosted antibody that specifically recognizes human histone H4, particularly around the lysine 79 residue. This antibody has been validated for multiple applications including ELISA, immunohistochemistry (IHC), and chromatin immunoprecipitation (ChIP). The immunogen used for its production is a peptide sequence derived from Human Histone H4 around the site of Lys (79). Its target protein is identified by accession number P62805, and it recognizes numerous histone H4 variants and subtypes .

While most histone H4 antibodies target the N-terminal tail where many post-translational modifications occur, this antibody targets a region around Lys-79, providing researchers with a tool to study this specific region of the histone. The polyclonal nature of this antibody means it can recognize multiple epitopes on the target protein, potentially providing stronger signals in certain applications.

What applications has HIST1H4A (Ab-79) Antibody been validated for?

The HIST1H4A (Ab-79) antibody has been specifically validated for three primary applications:

• ELISA (Enzyme-Linked Immunosorbent Assay): For quantitative detection of histone H4 in solution

• IHC (Immunohistochemistry): For visualization of histone H4 in tissue sections

• ChIP (Chromatin Immunoprecipitation): For studying protein-DNA interactions involving histone H4

Each application requires specific optimization parameters. For instance, when using this antibody for ChIP assays, researchers have successfully employed it to study the relationship between histone H4 acetylation and chromatin binding of other proteins such as IκBα, as demonstrated in studies with HCT-116 colorectal cancer cells .

How should HIST1H4A (Ab-79) Antibody be stored and handled to maintain its efficacy?

Based on standard practices for similar antibodies, the HIST1H4A (Ab-79) antibody should be stored according to the following guidelines to maintain optimal activity:

• Store at -20°C for long-term storage

• Avoid repeated freeze-thaw cycles which may denature the antibody

• Store in small aliquots to minimize freeze-thaw cycles

• For short-term storage (1-2 weeks), the antibody can be kept at 4°C

• Frost-free freezers are not recommended for storage

When handling the antibody, researchers should work aseptically to prevent contamination, which can introduce variables into experimental results. Additionally, before use, the antibody should be gently mixed but not vortexed to avoid protein denaturation.

How can HIST1H4A (Ab-79) Antibody be optimized for ChIP assays investigating histone modifications?

For optimal ChIP performance with HIST1H4A (Ab-79) antibody, researchers should consider the following methodological approach:

Analysis of ChIP data from HCT-116 colorectal cancer cells demonstrated that the antibody could successfully identify enrichment of histone H4 at specific genomic regions, particularly showing overlap with acetylated histone marks at transcription start sites (TSS) .

What is the relationship between histone H4 acetylation and tissue stem cell compartments as revealed by immunohistochemistry?

Research using histone H4 antibodies has revealed fascinating biological patterns related to histone modifications in different tissue compartments:

Immunohistochemistry studies have demonstrated that acetylated histone H4 species (including H4K12Ac) are predominantly localized in stem cell compartments of tissues such as intestinal crypts and the basal layer of skin. This contrasts with methylated forms like H4K20me2,3, which are primarily found in differentiated cells .

Specifically:

• Antibodies against acetylated histone H4 labeled cells localized in the intestinal crypt compartment, colocalizing with p-IκBα and including the canonical Lgr5+ intestinal stem cells.

• H4K12Ac was primarily detected in keratinocytes of the basal layer of skin and hair follicles, where progenitors and stem cells reside.

• In contrast, H4K20me2,3 was exclusively present in differentiated cells of the mouse intestinal villi and skin.

• This compartmentalization of histone H4 modifications progressively develops during embryonic development .

These findings suggest that HIST1H4A (Ab-79) antibody could be valuable for studying stem cell biology and differentiation processes through the lens of histone H4 modifications.

How can HIST1H4A (Ab-79) Antibody be used to study histone H4 dynamics during cell differentiation?

Western blotting analyses with histone H4 antibodies have revealed important insights into the dynamics of histone H4 modifications during cell differentiation:

• Temporal dynamics: Studies in HT29 colorectal cancer cells showed that H4K12 acetylation reaches maximum levels around days 3-5 of post-confluence, coinciding with the peak binding of IκBα and p-IκBα to chromatin.

• Histone cleavage events: Histone H4 cleavage, as detected with H4K20me2,3 antibody, begins early at post-confluence and reaches maximum at day 5, correlating with cell differentiation.

• Functional impact: IκBα deletion prevented goblet cell differentiation of HT29 cells, suggesting a functional relationship between chromatin-associated IκBα and histone H4 modifications during differentiation .

To study similar dynamics using HIST1H4A (Ab-79) antibody, researchers should:

• Establish a time course experiment with appropriate differentiation model

• Collect samples at regular intervals

• Perform parallel analyses of histone H4 status and differentiation markers

• Use complementary antibodies targeting different histone H4 modifications

This approach can provide insights into how histone H4 and its modifications contribute to cell fate decisions and differentiation processes.

What are the most effective protocols for dual immunostaining with HIST1H4A (Ab-79) Antibody and other histone modification markers?

For effective dual immunostaining experiments with HIST1H4A (Ab-79) antibody and other histone modification markers, researchers should consider the following protocol:

• Fixation optimization:

  • For frozen sections: Acetone fixation for 10 minutes at -20°C

  • For paraffin sections: 4% paraformaldehyde followed by antigen retrieval

  • Note that acetone or PFA fixation is recommended for optimal results with histone antibodies

• Blocking strategy:

  • Use 5-10% normal serum from the species of the secondary antibody

  • Include 0.1-0.3% Triton X-100 for nuclear penetration

  • Consider adding 1% BSA to reduce non-specific binding

• Primary antibody incubation:

  • Begin with a 1:100-1:500 dilution of HIST1H4A (Ab-79) antibody

  • For dual staining, select complementary antibodies from different host species

  • Incubate overnight at 4°C for optimal binding

• Secondary antibody selection:

  • Use fluorophore-conjugated secondary antibodies with minimal spectral overlap

  • For rabbit-hosted antibodies like HIST1H4A (Ab-79), pair with mouse-hosted antibodies (e.g., H4K12ac or H4K20me2,3) for dual staining

• Controls:

  • Single antibody controls

  • Secondary-only controls

  • Isotype controls

This approach has been successfully used in studies examining the co-localization of histone H4 modifications with other proteins such as p-IκBα in intestinal crypt cells and skin tissue sections .

What is the recommended panel of histone modification antibodies to use alongside HIST1H4A (Ab-79) for comprehensive epigenetic profiling?

Based on research literature, a comprehensive panel for epigenetic profiling alongside HIST1H4A (Ab-79) antibody should include:

This panel provides a balance of acetylation and methylation marks, allowing researchers to distinguish between active and repressive chromatin states. Studies have shown that these modifications exhibit distinct patterns in different cell types and developmental stages. For example, acetylation marks (H4K5ac, H4K8ac, H4K12ac, H4K16ac) are enriched in stem cell compartments, while methylation marks like H4K20me2,3 are predominantly found in differentiated cells .

What are common challenges when using HIST1H4A (Ab-79) Antibody in ChIP assays and how can they be addressed?

Researchers often encounter several challenges when performing ChIP assays with histone antibodies like HIST1H4A (Ab-79). Here are solutions to common issues:

• High background signal:

  • Increase washing stringency with higher salt concentrations

  • Pre-clear chromatin with protein A/G beads before antibody addition

  • Increase blocking reagent concentration

  • Use a more specific secondary antibody

• Low signal-to-noise ratio:

  • Optimize crosslinking time (typically 10-15 minutes for histone studies)

  • Improve sonication efficiency to generate 200-500bp fragments

  • Increase antibody concentration incrementally

  • Extend antibody incubation time to overnight at 4°C

• Poor enrichment of target regions:

  • Verify antibody specificity with western blot before ChIP

  • Confirm that the epitope is accessible in crosslinked chromatin

  • Test different chromatin preparation methods

  • Include positive control regions known to contain histone H4

• Inconsistent results between replicates:

  • Standardize cell culture conditions

  • Prepare a single batch of crosslinked chromatin for all replicates

  • Use automation where possible to reduce technical variation

  • Implement rigorous quality control for each step

Studies analyzing histone H4 acetylation in HCT-116 cells successfully addressed these challenges by optimizing antibody selection and chromatin preparation, resulting in significant enrichment of IκBα peaks in acetylated chromatin regions with a p-value < 0.01 compared to random distribution .

How can researchers validate the specificity of HIST1H4A (Ab-79) Antibody for their particular experimental system?

Validating antibody specificity is critical for generating reliable data. For HIST1H4A (Ab-79) antibody, researchers should implement these validation strategies:

• Western blot analysis:

  • Run nuclear extracts alongside recombinant histone H4

  • Confirm single band at approximately 11 kDa (histone H4 molecular weight)

  • Include other histone proteins (H2A, H2B, H3) as negative controls

  • Test with extracts from multiple cell types

• Peptide competition assay:

  • Pre-incubate antibody with excess immunizing peptide

  • Compare binding with and without peptide competition

  • Specific binding should be significantly reduced in the presence of peptide

• Knockout/knockdown validation:

  • Test antibody in cells with HIST1H4A knockdown

  • Compare signals between wild-type and knockdown samples

  • Specific antibodies should show reduced signal in knockdown cells

• Cross-reactivity testing:

  • Test against related histone variants

  • Evaluate detection in multiple species if cross-reactivity is claimed

  • Ensure antibody doesn't recognize post-translational modifications unintentionally

Similar histone antibodies have demonstrated their specificity through these methods. For example, the Histone H4 (D2X4V) Rabbit mAb has been validated to recognize endogenous levels of total histone H4 protein without cross-reacting with other histone proteins , providing a model for proper validation procedures.

How does HIST1H4A (Ab-79) Antibody performance compare with other commercial histone H4 antibodies in different applications?

When selecting a histone H4 antibody, researchers should consider the comparative performance across different applications:

The choice between these antibodies depends on the specific research question:

• For studying total histone H4 levels regardless of modifications, Histone H4 (D2X4V) antibody offers high specificity with no cross-reactivity .

• For investigating specific acetylation patterns, H4K12ac antibody has been successfully used to identify stem cell compartments in tissues .

• HIST1H4A (Ab-79) antibody's unique targeting of the region around Lys-79 makes it valuable for studying specific aspects of histone H4 biology .

Research has demonstrated that antibodies targeting different histone H4 epitopes can reveal distinct biological patterns, such as the compartmentalization of acetylated H4 in stem cells and methylated H4 in differentiated cells .

What insights have been gained from using histone H4 antibodies in understanding the relationship between chromatin modifications and cell differentiation?

Research using histone H4 antibodies has provided significant insights into chromatin dynamics during cell differentiation:

• Temporal dynamics of histone modifications:

  • Western blot analyses of chromatin extracts from HT29 colorectal cancer cells showed that H4K12 acetylation levels change during cell differentiation

  • H4K12 acetylation reaches maximum around days 3-5 post-confluence

  • Histone H4 cleavage begins early at post-confluence and reaches maximum at day 5

• Spatial distribution in tissues:

  • Acetylated histone H4 species are predominantly localized in stem cell compartments

  • H4K12Ac is primarily detected in the basal layer of skin and hair follicles

  • H4K20me2,3 is exclusively present in differentiated cells of intestinal villi and skin

  • This compartmentalization develops progressively during embryonic development

• Functional significance:

  • IκBα binding to chromatin parallels H4K12 acetylation patterns

  • IκBα deletion precluded goblet cell differentiation of human HT29 CRC cells

  • This suggests a functional link between chromatin modifications, transcription factor binding, and cell differentiation

These findings highlight the importance of histone H4 and its modifications in regulating cell fate decisions and tissue homeostasis, providing valuable research directions for investigators using HIST1H4A (Ab-79) antibody.

What emerging applications of histone H4 antibodies like HIST1H4A (Ab-79) are being developed in cancer research and regenerative medicine?

Histone H4 antibodies are increasingly being utilized in cutting-edge research applications:

• Single-cell epigenomics:

  • Combining histone H4 antibodies with single-cell technologies to map epigenetic heterogeneity within tumors

  • Identifying rare cell populations with distinct histone modification profiles

  • Correlating these profiles with tumor progression and treatment resistance

• Liquid biopsy development:

  • Detecting circulating histone H4 and its modified forms as potential biomarkers

  • Using histone H4 modification patterns to monitor treatment response

  • Developing minimally invasive diagnostic tools based on histone profiles

• Regenerative medicine applications:

  • Mapping histone H4 modification changes during stem cell differentiation

  • Engineering epigenetic states to direct cell fate decisions

  • Creating defined culture conditions that maintain specific histone modification patterns

• Therapeutic targeting:

  • Developing drugs that target writers, readers, and erasers of histone H4 modifications

  • Using histone H4 antibodies to monitor drug efficacy

  • Combining epigenetic therapies with conventional treatments

Studies have already demonstrated the value of histone H4 antibodies in understanding the relationship between epigenetic modifications and stem cell compartments , providing a foundation for these emerging applications.