HIST1H4A (Ab-20) Antibody

What is HIST1H4A and what is its role in chromatin biology?

HIST1H4A (Histone Cluster 1, H4a) is a core component of the nucleosome, the fundamental unit of chromatin structure in eukaryotic cells . Nucleosomes wrap and compact DNA, limiting its accessibility to cellular machinery that requires DNA as a template for processes like transcription and replication . As a histone protein, HIST1H4A plays a central role in transcription regulation, DNA repair, DNA replication, and chromosomal stability . The protein's function is regulated through post-translational modifications (PTMs), which collectively contribute to the "histone code" that determines chromatin state and gene expression patterns . These modifications, particularly acetylation at specific lysine residues, create binding sites for regulatory proteins and affect the physical properties of chromatin, influencing DNA accessibility and gene expression .

What are the key characteristics of the HIST1H4A (Ab-20) Antibody?

The HIST1H4A (Ab-20) Antibody is a polyclonal antibody raised in rabbits that specifically recognizes histone H4 acetylated at lysine 20 (acLys20) . This antibody is primarily intended for research applications including Immunofluorescence (IF), Chromatin Immunoprecipitation (ChIP), Enzyme-Linked Immunosorbent Assay (ELISA), and Immunohistochemistry (IHC) . It demonstrates reactivity with human samples, making it valuable for studying human cell lines and tissues . The antibody is supplied in liquid form and is unconjugated, allowing researchers flexibility in designing detection systems appropriate for their specific experimental needs . The antibody recognizes the H4 clustered histone 9 (H4C9) and has been validated for detecting the protein with ID P62805 (Histone H4) .

How does HIST1H4A (Ab-20) Antibody compare to other histone modification antibodies?

HIST1H4A (Ab-20) Antibody is part of a broader collection of antibodies targeting various modifications on histone H4, each with distinct specificities and applications . While the Ab-20 antibody specifically recognizes acetylation at lysine 20, other antibodies target different modifications including acetylation at lysine residues 8, 12, 16, 31, 56, and 79, as well as methylation at lysine 20 . These different antibodies enable researchers to investigate the diverse array of post-translational modifications that constitute the histone code . The specificity of each antibody for its corresponding modification is critical for accurate experimental results, particularly in applications like ChIP where precise identification of modified histones at specific genomic locations is essential . When selecting among these antibodies, researchers should carefully consider the specific modification they wish to study and ensure the antibody has been validated for their intended application .

What are the validated research applications for HIST1H4A (Ab-20) Antibody?

The HIST1H4A (Ab-20) Antibody has been validated for multiple research applications, providing versatility across different experimental methodologies . For chromatin studies, it has been specifically validated for Chromatin Immunoprecipitation (ChIP), enabling the mapping of histone H4K20 acetylation patterns across the genome . In cellular localization studies, the antibody has been validated for Immunofluorescence (IF) and Immunocytochemistry (ICC), allowing visualization of H4K20ac distribution within cellular compartments . For protein detection, it has been validated for Enzyme-Linked Immunosorbent Assay (ELISA), offering a sensitive method for quantifying H4K20ac levels in various samples . Additionally, the antibody has been validated for Immunohistochemistry (IHC), as demonstrated by successful staining in paraffin-embedded human cervical cancer tissue using a Leica BondTM system . This broad range of validated applications makes the antibody a versatile tool for researchers investigating histone modifications in diverse experimental contexts.

How should immunohistochemistry (IHC) protocols be optimized for HIST1H4A (Ab-20) Antibody?

Optimizing immunohistochemistry protocols for HIST1H4A (Ab-20) Antibody requires careful attention to several critical parameters . Based on validated protocols, researchers should begin with dewaxing and hydration of paraffin-embedded tissue sections, followed by antigen retrieval mediated by high pressure in citrate buffer (pH 6.0) . Sections should be blocked with 10% normal goat serum for 30 minutes at room temperature to reduce non-specific binding . The primary antibody should be diluted to 1:100 in 1% BSA and incubated at 4°C overnight to ensure optimal binding . Detection can be achieved using a biotinylated secondary antibody, visualized with an HRP-conjugated SP system . For troubleshooting common IHC issues, researchers should consider adjusting antibody concentration, incubation time, or antigen retrieval conditions if staining is too weak or shows high background . Positive and negative controls should always be included to validate staining specificity and protocol efficacy .

What considerations are important for chromatin immunoprecipitation (ChIP) using HIST1H4A (Ab-20) Antibody?

When performing chromatin immunoprecipitation with HIST1H4A (Ab-20) Antibody, several technical considerations should be addressed to ensure experimental success . First, chromatin preparation is critical—researchers should optimize crosslinking conditions (typically 1% formaldehyde for 10 minutes) and sonication parameters to generate chromatin fragments of 200-500bp . For the immunoprecipitation step, the antibody concentration should be carefully titrated, with 2-5μg typically sufficient for standard ChIP experiments . Researchers should include appropriate controls, such as IgG negative controls and positive controls using antibodies against abundant histone marks . For ChIP-seq applications, the antibody has been specifically validated, indicating its suitability for genome-wide mapping of H4K20ac distribution . When analyzing ChIP data, researchers should consider the broader chromatin context, as H4K20 acetylation often occurs within specific genomic regions and may correlate with other histone modifications that influence transcriptional activity . Cross-reactivity testing with related modifications (e.g., other acetylated lysines on H4) is advisable to confirm specificity when interpreting ChIP results .

How is HIST1H4A-IgG used in diagnostic applications for autoimmune diseases?

HIST1H4A-IgG has emerged as a highly promising biomarker for systemic lupus erythematosus (SLE), demonstrating exceptional diagnostic performance that significantly enhances current diagnostic approaches . In a comprehensive study involving 153 SLE patients and 81 healthy controls, HIST1H4A-IgG demonstrated the best individual diagnostic performance among tested markers, with an area under the curve (AUC) of 0.97 and sensitivity of 95% at 90% specificity . The diagnostic utility extends beyond simple detection, as HIST1H4A-IgG was identified as an independent significant predictor for SLE diagnosis in multivariate modeling (P < 0.0001) . Importantly, when combined with standard antibody parameters (including SSA, SSB, Sm, U1-RNP, and RPLP2), HIST1H4A-IgG significantly improved prediction of SLE over standard parameters alone (residual deviance 45.9 vs 97.1, P = 4.3 × 10-11) . This improved the diagnostic accuracy from 86% to 89% in validation samples . These findings position HIST1H4A-IgG as a valuable addition to the diagnostic toolkit for autoimmune disease, particularly for SLE, where early and accurate diagnosis remains challenging.

What is the relationship between HIST1H4A acetylation patterns and gene expression regulation?

The acetylation of HIST1H4A at lysine 20 (H4K20ac) represents a specific epigenetic modification with important implications for gene expression regulation . This modification typically correlates with transcriptionally active chromatin regions, as the addition of acetyl groups neutralizes the positive charge of lysine residues, reducing the electrostatic interaction between histones and DNA and promoting a more open chromatin structure . In the context of the histone code, H4K20ac frequently co-occurs with other active histone marks such as H3K4me3 and H3K27ac, collectively creating binding platforms for transcriptional activators and chromatin remodeling complexes . The specific genomic distribution of H4K20ac often shows enrichment at promoters and enhancers of actively transcribed genes, suggesting its role in facilitating transcription initiation and elongation . Understanding the precise relationship between H4K20ac and gene expression requires integrated analysis of ChIP-seq data with transcriptomic profiles, which can reveal gene sets whose expression correlates with this specific histone modification . Advanced research techniques combining HIST1H4A (Ab-20) Antibody with other epigenetic profiling methods can provide comprehensive insights into the functional significance of this modification in various cellular contexts and disease states .

What experimental approaches can detect dynamic changes in HIST1H4A modifications during cellular processes?

Investigating dynamic changes in HIST1H4A modifications during cellular processes requires sophisticated experimental approaches that capture temporal and spatial dynamics . Time-course ChIP-seq experiments using HIST1H4A (Ab-20) Antibody can map genome-wide changes in H4K20ac distribution during processes like cell differentiation, cell cycle progression, or response to environmental stimuli . For cellular localization dynamics, live-cell imaging techniques can be combined with fixed-cell immunofluorescence using the antibody at different time points to visualize redistribution of H4K20ac within the nucleus . Mass spectrometry-based approaches provide complementary quantitative data on changes in H4K20ac abundance relative to other histone modifications, offering insights into the broader epigenetic landscape changes . Single-cell techniques represent the cutting edge in this field, with single-cell ChIP-seq or CUT&Tag approaches using HIST1H4A (Ab-20) Antibody potentially revealing cell-to-cell heterogeneity in H4K20ac patterns during cellular transitions . For functional validation, researchers can combine these approaches with genetic or pharmacological manipulation of histone acetyltransferases and deacetylases that target H4K20, correlating changes in acetylation with phenotypic outcomes .

What are common challenges in Western blotting with HIST1H4A (Ab-20) Antibody and how can they be addressed?

Western blotting with HIST1H4A (Ab-20) Antibody presents several technical challenges that require specific troubleshooting approaches . One common issue is weak or absent signals, which may result from insufficient protein loading or inefficient transfer of low molecular weight histone proteins (~11 kDa for H4) . This can be addressed by using specialized transfer conditions optimized for small proteins, including shorter transfer times or lower methanol concentrations in transfer buffer . Another challenge is high background, which can be mitigated by increasing blocking time (2-3 hours) with 5% BSA instead of milk proteins, which may contain endogenous phosphorylation that cross-reacts with the antibody . For improved specificity, researchers should consider using PVDF membranes instead of nitrocellulose and optimizing primary antibody dilution and incubation conditions (1:1000 dilution, overnight at 4°C is recommended) . When multiple bands appear, this may indicate cross-reactivity with other acetylated histones; including a blocking peptide control can help confirm specificity . Additionally, sample preparation is critical—histone extraction protocols using acid extraction methods are preferred over standard RIPA buffer extraction to enrich for histone proteins and improve detection sensitivity .

How can researchers verify the specificity of HIST1H4A (Ab-20) Antibody for acetylated lysine 20?

Verifying the specificity of HIST1H4A (Ab-20) Antibody for acetylated lysine 20 is essential for reliable experimental results and can be accomplished through several complementary approaches . Peptide competition assays provide a direct test of specificity—pre-incubating the antibody with synthetic peptides containing acetylated K20 should abolish signal, while pre-incubation with unmodified peptides or peptides acetylated at other lysine residues should not affect binding . For more comprehensive validation, researchers can use dot blot or peptide array analysis with a panel of differentially modified histone peptides to assess potential cross-reactivity with similar modifications (e.g., acetylation at K16 or K12) . Genetic approaches provide additional validation—using cell lines with CRISPR-engineered K20R mutations (preventing acetylation at this site) should eliminate antibody binding . Pharmacological approaches offer further confirmation—treating cells with histone deacetylase inhibitors should increase the signal, while treatment with specific acetyltransferase inhibitors targeting H4K20 should decrease it . Mass spectrometry analysis of immunoprecipitated histones can provide definitive evidence of antibody specificity by confirming the presence of acetylated K20 in the immunoprecipitated material . Together, these validation strategies ensure confidence in the specificity of the antibody for its intended target .

What quality control parameters should researchers evaluate before using HIST1H4A (Ab-20) Antibody in critical experiments?

Before employing HIST1H4A (Ab-20) Antibody in critical experiments, researchers should conduct thorough quality control assessments to ensure reliable results . Lot-to-lot consistency should be evaluated by comparing performance across different antibody lots using standard samples and assays, as manufacturing variations can affect specificity and sensitivity . Researchers should verify application-specific performance by testing the antibody in the specific experimental context intended for use, as antibodies may perform differently across applications like ChIP, Western blotting, or immunofluorescence . Cross-reactivity testing with closely related modifications (especially other acetylated lysines on H4) should be conducted using modified peptide arrays or dot blots to confirm specificity . Stability assessment is also important—monitoring antibody performance over time and under different storage conditions can identify potential degradation issues . Positive and negative controls should be included in each experiment—cell lines or tissues known to express high levels of H4K20ac versus those with low or absent levels (e.g., after treatment with specific deacetylase inhibitors) . For quantitative applications, standard curves using recombinant acetylated histones can help determine the linear range of detection and sensitivity limits . Documentation of these quality control parameters provides critical assurance of experimental reliability and reproducibility .

How is HIST1H4A (Ab-20) Antibody being used in cancer research?

HIST1H4A (Ab-20) Antibody is finding increasing application in cancer research, particularly in investigating epigenetic alterations that contribute to tumorigenesis and cancer progression . The antibody has been successfully used for immunohistochemical analysis of human cervical cancer tissue, enabling the visualization and quantification of H4K20ac patterns in tumor samples . This application provides valuable insights into the epigenetic landscape of cancer cells, potentially identifying altered histone modification patterns that contribute to dysregulated gene expression in malignant transformation . By combining HIST1H4A (Ab-20) Antibody-based ChIP-seq with transcriptomic analysis, researchers can identify genes whose expression correlates with altered H4K20ac patterns in cancer, potentially revealing novel oncogenic pathways and therapeutic targets . The antibody's utility extends to monitoring epigenetic changes in response to cancer therapies, particularly those targeting histone modifying enzymes like histone deacetylase inhibitors, which are increasingly important in precision oncology approaches . As cancer epigenetics continues to emerge as a critical area for understanding tumor biology and developing targeted therapies, HIST1H4A (Ab-20) Antibody represents an important tool for investigating the specific contribution of H4K20 acetylation to cancer pathogenesis and treatment response .

What emerging technologies are enhancing the utility of HIST1H4A (Ab-20) Antibody in epigenetic research?

Emerging technologies are significantly expanding the utility of HIST1H4A (Ab-20) Antibody in epigenetic research, enabling more sophisticated investigations of H4K20 acetylation dynamics and function . CUT&Tag and CUT&RUN technologies represent advances over traditional ChIP, offering improved signal-to-noise ratios and requiring fewer cells, making them particularly valuable for analyzing rare cell populations or clinical samples . Single-cell epigenomic approaches are revolutionizing the field by allowing HIST1H4A (Ab-20) Antibody to be used for mapping H4K20ac distributions at single-cell resolution, revealing heterogeneity in epigenetic states that would be masked in bulk population analyses . Spatial technologies like imaging mass cytometry combined with HIST1H4A (Ab-20) Antibody immunostaining enable visualization of H4K20ac patterns within their tissue context, preserving spatial information critical for understanding epigenetic regulation in complex tissues . Computational advancements in integrative multi-omics analyses allow researchers to correlate H4K20ac patterns detected by the antibody with other epigenetic marks, transcriptomic data, and phenotypic outcomes, providing comprehensive insights into regulatory networks . CRISPR-based epigenome editing technologies complement antibody-based detection by enabling targeted manipulation of H4K20 acetylation at specific genomic loci, followed by detection with HIST1H4A (Ab-20) Antibody to confirm modification and investigate functional consequences .

What is the significance of HIST1H4A-IgG as a biomarker in clinical research?

The emergence of HIST1H4A-IgG as a biomarker represents a significant advancement in clinical research, particularly for autoimmune diseases like systemic lupus erythematosus (SLE) . The exceptional diagnostic performance of HIST1H4A-IgG, with an area under the curve of 0.97 and sensitivity of 95% at 90% specificity, positions it as one of the most accurate individual biomarkers for SLE identification . This level of performance has important implications for patient stratification in clinical trials, potentially enabling more precise selection of participants based on specific autoantibody profiles . Beyond diagnosis, longitudinal monitoring of HIST1H4A-IgG levels may provide insights into disease activity and treatment response, though additional research is needed to establish its utility as a monitoring biomarker . The combination of HIST1H4A-IgG with standard antibody markers significantly improves diagnostic accuracy from 86% to 89% in validation samples, suggesting that multiplex panel approaches incorporating this marker could enhance clinical decision-making . From a pathophysiological perspective, the presence of autoantibodies against histones provides insights into the mechanisms of immune dysregulation in SLE, potentially identifying subgroups of patients with distinct immunopathological features . As personalized medicine approaches gain traction in autoimmune disease management, HIST1H4A-IgG may contribute to more tailored treatment strategies based on specific autoantibody signatures .