2-hydroxyisobutyryl-HIST1H4A (K16) Antibody

What is the 2-hydroxyisobutyryl-HIST1H4A (K16) Antibody and what does it detect?

The 2-hydroxyisobutyryl-HIST1H4A (K16) Antibody is a polyclonal antibody that specifically recognizes the 2-hydroxyisobutyryl modification at lysine 16 position on histone H4. This post-translational modification is part of the broader family of histone acylations that regulate chromatin structure and gene expression. The antibody targets the peptide sequence surrounding the 2-hydroxyisobutyryl-Lys (16) site derived from Human Histone H4, providing researchers with a specific tool to investigate this epigenetic mark . The antibody enables detection of this modification which plays critical roles in transcriptional regulation through altering chromatin accessibility.

What experimental applications has the 2-hydroxyisobutyryl-HIST1H4A (K16) Antibody been validated for?

The 2-hydroxyisobutyryl-HIST1H4A (K16) Polyclonal Antibody has been validated for multiple experimental applications crucial to epigenetic research. These validated applications include Enzyme-Linked Immunosorbent Assay (ELISA), Western Blotting (WB), Immunofluorescence (IF), Immunoprecipitation (IP), and Chromatin Immunoprecipitation (ChIP) . This versatility makes it particularly valuable for researchers investigating histone modifications through complementary techniques. Each application provides different insights: Western Blotting confirms the presence and quantity of the modification, Immunofluorescence reveals cellular localization patterns, while ChIP assays determine genomic binding sites of the modified histones, allowing for comprehensive characterization of this epigenetic mark.

How does 2-hydroxyisobutyrylation at H4K16 compare with other histone post-translational modifications?

2-hydroxyisobutyrylation at H4K16 represents a distinct histone modification that differs from other post-translational modifications in several key aspects. Unlike more well-characterized modifications such as acetylation, 2-hydroxyisobutyrylation incorporates a bulkier chemical group that may exert stronger effects on chromatin accessibility. Research in plants has shown that histone Khib sites generally do not overlap with frequently modified N-tail lysines such as H3K4, H3K9, and H4K8, suggesting unique genomic targeting . Interestingly, histone Khib shows high correlation with acetylation marks, particularly H3K23ac, in terms of genomic and genic distribution patterns . Their co-enrichment correlates strongly with high gene expression levels, indicating potentially synergistic roles in transcriptional activation. This relationship suggests that Khib may work in concert with acetylation to regulate gene expression through complementary mechanisms.

What are the recommended storage conditions and handling protocols for the 2-hydroxyisobutyryl-HIST1H4A (K16) Antibody?

For optimal antibody performance, the 2-hydroxyisobutyryl-HIST1H4A (K16) Antibody should be stored at -20°C or -80°C immediately upon receipt. Repeated freeze-thaw cycles should be strictly avoided as they significantly compromise antibody functionality and specificity . Working aliquots can be prepared and stored at 4°C for up to one week, while long-term storage requires freezing conditions. The antibody is typically supplied in a liquid form with a stabilizing buffer containing 50% glycerol and 0.01M PBS at pH 7.4, with 0.03% Proclin 300 added as a preservative . When handling the antibody, researchers should wear appropriate personal protective equipment, maintain sterile conditions to prevent contamination, and follow institution-specific guidelines for biological materials. Proper storage and handling ensure maximum antibody sensitivity and reproducibility across experiments.

What are the optimal conditions for using 2-hydroxyisobutyryl-HIST1H4A (K16) Antibody in ChIP-seq experiments?

Optimizing ChIP-seq experiments with the 2-hydroxyisobutyryl-HIST1H4A (K16) Antibody requires careful attention to several critical parameters. For chromatin preparation, formaldehyde crosslinking should be performed at 1% concentration for 10 minutes at room temperature, followed by quenching with glycine. Sonication conditions must be empirically determined to achieve chromatin fragments between 200-500 bp, with fragment size verification by gel electrophoresis. The antibody-to-chromatin ratio typically requires 3-5 μg of antibody per 25 μg of chromatin, though optimization is recommended. Overnight incubation at 4°C with gentle rotation provides optimal antibody-antigen binding. Studies have shown that histone Khib is highly enriched at transcription start sites and primarily located in genic regions, with approximately 35% of protein-coding genes containing this modification . Including appropriate controls is crucial - IgG negative controls and positive controls targeting well-characterized histone marks help validate specificity. For downstream analysis, specialized peak-calling algorithms that account for the unique distribution pattern of Khib modifications are recommended for accurate data interpretation.

How can researchers differentiate between 2-hydroxyisobutyrylation and other acylation marks in complex histone modification studies?

Differentiating between 2-hydroxyisobutyrylation and other acylation marks requires a multi-faceted approach combining mass spectrometry, specific antibody validation, and comparative genomics. High-resolution mass spectrometry represents the gold standard for definitively identifying Khib modifications versus other acylations, as each modification produces characteristic mass shifts and fragmentation patterns. Researchers should employ a targeted mass spectrometry approach using multiple reaction monitoring (MRM) to distinguish between similar acylations. Antibody specificity confirmation through dot-blot assays against various modified peptides is essential to rule out cross-reactivity . When analyzing ChIP-seq data, researchers should conduct comparative analyses of Khib with other acylation marks like acetylation to identify overlapping and distinct genomic regions. Studies in Arabidopsis thaliana demonstrated distinct distribution patterns of Khib compared to other modifications, while showing correlation with H3K23ac . Sequential ChIP experiments (re-ChIP) combining antibodies against different modifications can help determine co-occurrence at specific loci. This integrated approach ensures reliable differentiation between various acylation marks in complex epigenomic landscapes.

What technical challenges might researchers encounter when using 2-hydroxyisobutyryl-HIST1H4A (K16) Antibody in different experimental systems?

Researchers using the 2-hydroxyisobutyryl-HIST1H4A (K16) Antibody across varied experimental systems may encounter several technical challenges that require strategic troubleshooting. Cross-reactivity with other histone modifications represents a significant concern, particularly with structurally similar acylations. Therefore, validation through dot-blot assays or peptide competition experiments is crucial for confirming specificity in each experimental context . Species-specific variations in histone sequences may affect antibody recognition despite the high conservation of histone H4. While the antibody is validated for human samples , researchers working with other species should perform preliminary validation studies. Cell type-specific differences in chromatin accessibility and histone modification levels may necessitate protocol adjustments, including optimization of cell lysis conditions, extraction buffers, and antibody concentrations. For fixed tissues, optimization of antigen retrieval methods is essential to expose the epitope while preserving tissue morphology. Another challenge involves accurately quantifying subtle changes in modification levels, which may require careful normalization strategies and sensitive detection methods. The dynamic nature of histone modifications also presents temporal sampling challenges, particularly for transient modifications that may be rapidly added or removed in response to cellular signals.

How do different fixation and extraction protocols affect the detection of 2-hydroxyisobutyryl modification at H4K16?

The detection of 2-hydroxyisobutyryl modification at H4K16 is significantly influenced by fixation and extraction protocols, requiring careful optimization to preserve epitope integrity while achieving efficient protein extraction. Formaldehyde fixation, commonly used in immunofluorescence and ChIP applications, can mask epitopes through extensive crosslinking if performed at high concentrations or extended durations. Optimal protocols typically use 1% formaldehyde for 10 minutes at room temperature. For tissue samples, perfusion-based fixation provides more uniform results than immersion methods. Extraction buffers containing sodium butyrate (5-10 mM) help inhibit histone deacetylases that might remove acylation marks during sample processing. The extraction protocol should include protease inhibitors and, critically, deacylase inhibitors such as nicotinamide (NAM) and trichostatin A (TSA) to prevent modification loss. Studies with similar histone acylations have shown that acid extraction methods (using 0.2N HCl or 0.4N H2SO4) generally preserve modifications better than salt extraction techniques. For immunofluorescence applications, heat-mediated antigen retrieval in citrate buffer (pH 6.0) typically yields optimal epitope accessibility. The dynamic nature of histone modifications necessitates rapid sample processing, with immediate flash freezing recommended for samples that cannot be processed immediately.

What is the relationship between 2-hydroxyisobutyrylation at H4K16 and gene expression patterns?

The relationship between 2-hydroxyisobutyrylation at H4K16 and gene expression represents a critical aspect of epigenetic regulation. Chromatin immunoprecipitation sequencing (ChIP-seq) studies have demonstrated that histone Khib modifications are predominantly located in genic regions, with significant enrichment at transcription start sites (TSS) . This pattern suggests a functional role in gene activation. Notably, genomic co-enrichment of histone Khib and histone acetylation, particularly H3K23ac, strongly correlates with high gene expression levels . This correlation indicates a potential synergistic mechanism where multiple histone modifications collectively establish a transcriptionally permissive chromatin environment. Experimental evidence from plant models reveals that Khib marks concentrate on genes involved in fundamental metabolic processes, including starch and sucrose metabolism, pentose and glucuronate interconversions, and phenylpropanoid biosynthesis . This targeting pattern suggests a specialized role in regulating cellular metabolism. The functional significance of this modification appears particularly pronounced during stress responses, as demonstrated by studies showing that histone Khib and H3K23ac collaborate to fine-tune plant responses to dark-induced starvation . The presence of both modifications appears to promote high transcriptional activity, enabling adaptive metabolic adjustments during environmental challenges.

How does the distribution of 2-hydroxyisobutyrylation at H4K16 compare across different cell types and developmental stages?

The distribution patterns of 2-hydroxyisobutyrylation at H4K16 exhibit notable variability across cell types and developmental stages, reflecting tissue-specific epigenetic regulation. Although comprehensive comparative studies are still emerging, initial findings suggest both conserved and dynamic aspects of this modification. In plant models, Khib appears widely distributed across multiple species, including Arabidopsis, tobacco, rice, and maize, suggesting evolutionary conservation of this modification . Within cellular contexts, Khib modifications show enrichment patterns that correspond to specific functional gene categories, particularly those related to primary metabolism and stress responses . This targeted distribution indicates context-dependent regulation that may vary with cellular requirements. During developmental transitions, preliminary evidence suggests dynamic changes in Khib patterns, potentially serving as epigenetic switches that facilitate stage-specific gene expression programs. The temporal dynamics of Khib during cell differentiation likely involve coordinated activities of "writer" enzymes that incorporate the modification and "eraser" enzymes, such as HDA6 and HDA9 in plants, that remove it . Comparative analyses with other histone modifications reveal that Khib exhibits distribution patterns distinct from common histone marks like H3K4me3, H3K9me2, and H4K8ac, yet shows significant correlation with H3K23ac . This relationship suggests potential functional interactions between different histone modifications in orchestrating developmental gene expression programs.

What are the current hypotheses about the enzymatic machinery responsible for adding and removing 2-hydroxyisobutyryl modifications?

Current research has begun to elucidate the enzymatic machinery responsible for regulating 2-hydroxyisobutyrylation dynamics, though many aspects remain under investigation. For the addition ("writing") of Khib modifications, studies in mammalian systems have identified p300 and the MYST family acetyltransferase Tip60 as capable of catalyzing this modification . These enzymes demonstrate promiscuous activity toward different acyl-CoA cofactors, suggesting mechanistic overlap between various acylation pathways. The removal ("erasing") of Khib modifications in mammals involves histone deacetylases HDAC2 and HDAC3 . In plant systems, research has identified HDA6 and HDA9 as major candidates for Khib erasers in Arabidopsis . This finding indicates functional conservation of deacylase activities across eukaryotic kingdoms. The regulatory mechanisms controlling the activity of these enzymes likely involve metabolic signaling, as 2-hydroxyisobutyryl-CoA levels can influence modification rates. Current hypotheses suggest that cellular metabolic status directly impacts histone 2-hydroxyisobutyrylation through substrate availability, potentially linking metabolic pathways with epigenetic regulation. The temporal and spatial regulation of these enzymatic activities remains an active area of investigation, with evidence pointing to stress-responsive modulation of both writer and eraser activities. Understanding the complete enzymatic machinery will require further research on potential 'reader' proteins that specifically recognize and bind to Khib modifications to mediate downstream biological functions.

What evidence exists for 2-hydroxyisobutyrylation at H4K16 playing roles in cellular metabolism and stress responses?

Substantial evidence supports the role of 2-hydroxyisobutyrylation at H4K16 in regulating cellular metabolism and mediating stress responses. Research in plant systems has revealed that histone Khib modifications, in coordination with H3K23ac, are enriched on genes involved in critical metabolic pathways, including starch and sucrose metabolism, pentose and glucuronate interconversions, and phenylpropanoid biosynthesis . This targeted enrichment pattern suggests a specialized function in metabolic regulation. During dark-induced starvation conditions in Arabidopsis, both histone Khib and H3K23ac modifications contribute to fine-tuning plant stress responses . Metabolic profiling and transcriptome analyses have demonstrated correlations between these histone modifications and the expression of key metabolic genes such as GRANULE BOUND STARCH SYNTHASE 1 (GBSS1), SUCROSE SYNTHASE 6 (SUS6), and multiple genes involved in phenylpropanoid biosynthesis . The dynamic nature of these modifications in response to stress conditions suggests an adaptive role in reprogramming cellular metabolism to enhance survival. The involvement of HDA6 and HDA9 as Khib erasers further supports a regulatory mechanism where removal of this modification helps modulate gene expression during stress . These findings collectively suggest that 2-hydroxyisobutyrylation functions as a conserved yet unique histone mark that collaborates with other epigenetic modifications to regulate cellular metabolism, facilitating organismal adaptation to environmental challenges.

What are the recommended protocols for validating the specificity of 2-hydroxyisobutyryl-HIST1H4A (K16) Antibody before experimental use?

Thorough validation of 2-hydroxyisobutyryl-HIST1H4A (K16) Antibody specificity is crucial before experimental application to ensure reliable and reproducible results. A comprehensive validation strategy should include multiple complementary approaches. Peptide competition assays represent a fundamental validation method, where pre-incubation of the antibody with increasing concentrations of the specific 2-hydroxyisobutyrylated K16 peptide should progressively diminish signal intensity. Dot-blot assays using a panel of modified and unmodified histone peptides help assess cross-reactivity with similar modifications like acetylation or other acylations . Western blotting with recombinant histones serves as an essential negative control - the antibody should not detect unmodified recombinant H4 proteins but should recognize the modified form in cellular extracts . For ChIP applications, researchers should perform sequential ChIP experiments combining the Khib antibody with antibodies against known neighboring modifications to verify target specificity. Mass spectrometry validation provides the highest confidence level by confirming the precise modification detected by the antibody. Additionally, testing the antibody in knockout/knockdown models of enzymes responsible for adding or removing the modification can provide functional validation. Published studies have successfully employed dot-blot assays and recombinant histone negative controls to confirm anti-Khib antibody specificity , establishing these as reliable validation approaches.

What methodological considerations should be taken into account when performing quantitative analysis of 2-hydroxyisobutyrylation levels?

Quantitative analysis of 2-hydroxyisobutyrylation levels requires careful methodological considerations to ensure accurate, reproducible measurements across different experimental conditions. Sample preparation represents a critical initial step, with rapid processing essential to prevent modification loss. Extraction buffers should include deacylase inhibitors such as nicotinamide (10 mM) and trichostatin A (1 μM) to preserve modification levels. For Western blot quantification, loading normalization using total histone H4 antibodies is essential, with linearity verification across the detection range. Signal intensity should be measured within the linear dynamic range of detection to ensure proportionality to modification abundance. Mass spectrometry-based quantification offers higher precision but requires specialized approaches like stable isotope labeling or label-free quantification with appropriate internal standards. ChIP-qPCR quantification demands careful primer design targeting regions of interest, with normalization to input chromatin and inclusion of IgG controls to account for background. ChIP-seq quantification requires specialized normalization strategies, such as spike-in controls with exogenous chromatin, to enable accurate cross-sample comparisons. Biological variability necessitates sufficient biological replicates (minimum three) with statistical analysis appropriate for the experimental design and data distribution. Inter-laboratory standardization would benefit from the development of reference materials with defined 2-hydroxyisobutyrylation levels. These methodological considerations collectively ensure reliable quantitative analysis of this important epigenetic modification.

How can researchers design multiplexed experiments to investigate the interplay between 2-hydroxyisobutyrylation and other histone modifications?

Designing multiplexed experiments to investigate the interplay between 2-hydroxyisobutyrylation and other histone modifications requires sophisticated methodological approaches that capture both co-occurrence patterns and functional relationships. Sequential ChIP (re-ChIP) represents a powerful technique where chromatin is immunoprecipitated with one modification-specific antibody (e.g., anti-Khib), followed by a second immunoprecipitation with an antibody against another modification (e.g., anti-H3K23ac). This approach directly identifies genomic regions containing both modifications simultaneously. Multicolor immunofluorescence microscopy using differentially labeled secondary antibodies allows visualization of modification co-localization at the nuclear level, though careful controls for antibody cross-reactivity are essential. Mass spectrometry-based histone analysis can detect combinatorial modification patterns on the same histone tail through techniques like middle-down or top-down proteomics, providing direct evidence of modification co-occurrence on individual molecules. Recent advances in CUT&RUN or CUT&Tag technologies allow for higher sensitivity multiplexed profiling with lower cell numbers than traditional ChIP. Computational integration of multiple single-modification ChIP-seq datasets can reveal genomic co-enrichment patterns, as demonstrated for Khib and H3K23ac in plants . Genetic manipulation of enzymes that add or remove specific modifications, followed by monitoring effects on other modifications, helps establish causal relationships between different marks. These complementary approaches collectively enable comprehensive investigation of the complex interplay between 2-hydroxyisobutyrylation and other epigenetic modifications in diverse biological contexts.

What are the emerging technologies that might advance our understanding of 2-hydroxyisobutyrylation dynamics?

Emerging technologies show tremendous promise for advancing our understanding of 2-hydroxyisobutyrylation dynamics across diverse biological contexts. Single-cell epigenomics techniques are being adapted to study histone modifications with unprecedented cellular resolution, potentially revealing cell-type-specific patterns of 2-hydroxyisobutyrylation currently masked in bulk analyses. These approaches include single-cell ChIP-seq and CUT&Tag methodologies that require minimal cellular input. Live-cell imaging of histone modifications represents another frontier, with the development of specific sensors for real-time visualization of 2-hydroxyisobutyrylation dynamics in living cells. These approaches might employ genetically encoded readers fused to fluorescent proteins or antibody-based detection systems. CRISPR-based epigenome editing technologies offer the ability to precisely add or remove 2-hydroxyisobutyrylation at specific genomic loci, enabling causal studies of its functional impacts on gene regulation. This could involve fusion of catalytic domains from writers or erasers to catalytically inactive Cas9. Multi-omics integration approaches combining transcriptomics, proteomics, metabolomics, and epigenomics data will provide comprehensive views of how 2-hydroxyisobutyrylation connects to broader cellular processes. Advanced computational modeling using machine learning algorithms may predict 2-hydroxyisobutyrylation sites and their functional consequences based on DNA sequence features and chromatin contexts. These technological advances collectively promise to transform our understanding of this epigenetic modification's dynamic regulation and biological significance.

What are the potential therapeutic implications of understanding 2-hydroxyisobutyrylation mechanisms in disease contexts?

Understanding 2-hydroxyisobutyrylation mechanisms holds significant potential therapeutic implications across multiple disease contexts, particularly where epigenetic dysregulation contributes to pathogenesis. In cancer biology, altered histone modification patterns represent hallmarks of many malignancies. Preliminary evidence suggesting connections between histone acylations and metabolic reprogramming in cancer cells identifies 2-hydroxyisobutyrylation as a potential therapeutic target. Developing small molecule inhibitors that specifically target enzymes responsible for adding or removing this modification could provide novel approaches for modulating gene expression in cancer cells. Neurodegenerative disorders frequently involve dysregulation of gene expression programs, with growing evidence implicating histone modifications in disease progression. Understanding 2-hydroxyisobutyrylation in neuronal contexts might reveal new intervention strategies for conditions like Alzheimer's or Parkinson's disease. Metabolic disorders represent another promising application area, given the demonstrated role of 2-hydroxyisobutyrylation in regulating genes involved in starch, sucrose, and phenylpropanoid metabolism in plant systems . Similar regulatory mechanisms may operate in human metabolic pathways, suggesting potential therapeutic targets for conditions like diabetes or obesity. The involvement of histone deacetylases such as HDAC2 and HDAC3 in removing Khib modifications points to potential repurposing of existing HDAC inhibitors for modulating 2-hydroxyisobutyrylation levels. Development of modification-specific drugs targeting the reading, writing, or erasing of 2-hydroxyisobutyrylation could enable precise modulation of this epigenetic mark with fewer off-target effects than current broader-spectrum epigenetic drugs.

How might cross-species comparative studies of 2-hydroxyisobutyrylation advance our understanding of its evolutionary conservation and functional significance?

Cross-species comparative studies of 2-hydroxyisobutyrylation offer a powerful approach to elucidate both the evolutionary conservation and functional significance of this histone modification across diverse organisms. Phylogenetic analysis of 2-hydroxyisobutyrylation patterns across evolutionary lineages would help identify conserved modification sites that likely serve fundamental cellular functions versus species-specific sites that might mediate specialized processes. Initial evidence already confirms the presence of histone Khib across multiple plant species including Arabidopsis, tobacco, rice, and maize, as well as in human cells, suggesting broad evolutionary conservation . Comparative genomics approaches examining the co-evolution of Khib sites with their target genes could reveal functional constraints and adaptive patterns across species. Structure-function analyses might uncover how 2-hydroxyisobutyrylation physically affects nucleosome stability and chromatin architecture in different species with varying genomic compositions. Functional studies comparing the effects of 2-hydroxyisobutyrylation on gene expression across species would help determine whether this modification serves consistent regulatory roles or has acquired specialized functions in different lineages. Metabolic comparisons are particularly relevant given evidence linking 2-hydroxyisobutyrylation to metabolic regulation in plants ; similar studies in other organisms could reveal whether this connection represents a universal feature or a lineage-specific adaptation. The enzymes responsible for adding and removing 2-hydroxyisobutyrylation also warrant comparative analysis, as studies have already identified both similarities and differences between plant and mammalian systems in terms of the deacylases involved . These multi-faceted comparative approaches would collectively advance our understanding of both the core conserved functions of 2-hydroxyisobutyrylation and its diverse adaptive roles across the tree of life.

What challenges remain in developing standardized protocols for studying 2-hydroxyisobutyrylation across different research contexts?

Despite significant advances in studying 2-hydroxyisobutyrylation, several challenges remain in developing standardized protocols applicable across diverse research contexts. Antibody standardization represents a primary concern, as different commercial antibodies may exhibit varying specificities and sensitivities, complicating cross-laboratory comparisons. The development of fully characterized, highly specific monoclonal antibodies with defined epitope binding properties would address this issue. Sample preparation variability significantly impacts modification detection, with differences in extraction methods, buffer compositions, and deacylase inhibitor usage all potentially affecting measured 2-hydroxyisobutyrylation levels. Establishing consensus protocols with standardized reagents would improve reproducibility. Quantification methodologies currently lack standardization, with Western blotting, mass spectrometry, and ChIP-based approaches each employing different normalization strategies and reporting units. Creating reference materials with defined 2-hydroxyisobutyrylation levels could provide calibration standards for quantitative assays. Data analysis pipelines for ChIP-seq and other genome-wide approaches require standardization, as different computational methods can yield varying results from identical datasets. The development of specialized algorithms optimized for detecting 2-hydroxyisobutyrylation patterns would enhance analytical consistency. Cell type and context dependencies introduce additional complexity, as modification patterns may vary substantially across different cellular backgrounds. Systematic characterization across a panel of standard cell lines or tissues would help contextualize results from specialized systems. Inter-laboratory proficiency testing through round-robin studies where multiple laboratories analyze identical samples would help identify and address methodological inconsistencies. Community-wide efforts to establish best practices, potentially through international working groups focused on histone acylation research, would accelerate progress toward standardized, robust protocols for studying this important epigenetic modification.

Technical Specifications of Available 2-hydroxyisobutyryl-HIST1H4A (K16) Antibodies

Comparative Analysis of 2-hydroxyisobutyrylation and Other Histone Modifications

Recommended Optimization Parameters for ChIP Experiments with 2-hydroxyisobutyryl-HIST1H4A (K16) Antibody

Genes Associated with 2-hydroxyisobutyrylation in Metabolic Regulation