2-hydroxyisobutyryl-HIST1H3A (K79) Antibody

What is lysine 2-hydroxyisobutyrylation and how does it differ from other histone modifications?

Lysine 2-hydroxyisobutyrylation (Khib) is a relatively recently identified post-translational modification that affects the association between histone and DNA. Unlike lysine acetylation, 2-hydroxyisobutyrylation is structurally and mechanistically distinct, showing quantitative responses to changes in glycolytic flux that differ from acetylation patterns . The modification involves the addition of a 2-hydroxyisobutyryl group to lysine residues in proteins, creating a larger and more hydrophobic modification than acetylation. This chemical difference likely contributes to its unique biological functions and regulatory mechanisms. Research indicates that many 2-hydroxyisobutyrylated sites are exclusive compared to other modifications like lysine acetylation and crotonylation, with 27 of 63 identified modified sites in human and mouse histones being unique to 2-hydroxyisobutyrylation . These structural and distribution differences suggest that Khib represents a functionally distinct class of epigenetic marks.

What is the significance of H3K79 2-hydroxyisobutyrylation specifically?

H3K79 (lysine 79 on histone H3) represents one of the most important sites for 2-hydroxyisobutyrylation with documented biological relevance. Studies have revealed that the stoichiometry of H3K79 2-hydroxyisobutyrylation in synchronized G2/M HeLa cells is comparable to or even higher than many histone acetylation marks with established biological functions . This high stoichiometry suggests that H3K79 2-hydroxyisobutyrylation likely plays significant roles in chromatin regulation during cell division and other cellular processes. The modification occurs in the globular domain of histone H3 rather than the N-terminal tail, potentially affecting nucleosome structure and stability in ways distinct from tail modifications. The dynamic nature of this modification during cell cycle progression further suggests its involvement in cell cycle-dependent gene regulation processes, making it a valuable target for epigenetic research.

How is 2-hydroxyisobutyryl-HIST1H3A (K79) Antibody generated and validated?

The 2-hydroxyisobutyryl-HIST1H3A (K79) Antibody is typically generated through immunization of host animals (commonly rabbits) with synthetic peptides containing the 2-hydroxyisobutyrylated lysine at position 79 of human histone H3.1 (HIST1H3A) . The antibody production process involves multiple purification steps, including antigen affinity purification to enhance specificity for the modified epitope. Validation of these antibodies typically involves several complementary approaches: (1) ELISA assays comparing binding to modified versus unmodified peptides, (2) immunofluorescence studies in cells with known 2-hydroxyisobutyrylation patterns, (3) Western blot analysis comparing wild-type and mutant H3K79 proteins, and (4) peptide competition assays to confirm specificity. Proper validation ensures that the antibody recognizes the specific 2-hydroxyisobutyrylated lysine residue rather than the same residue with different modifications or different residues with the same modification.

What are the primary research applications for 2-hydroxyisobutyryl-HIST1H3A (K79) Antibody?

The 2-hydroxyisobutyryl-HIST1H3A (K79) Antibody serves multiple critical applications in epigenetic research. Primary applications include chromatin immunoprecipitation followed by sequencing (ChIP-seq) to map the genomic distribution of H3K79 2-hydroxyisobutyrylation, providing insights into its relationship with gene regulation. The antibody is also valuable for immunofluorescence (IF) studies, as indicated in the product specifications (dilution range IF:1:50-1:200) , allowing visualization of the modification's nuclear distribution and potential colocalization with other chromatin marks. Additional applications include enzyme-linked immunosorbent assays (ELISA) for quantitative measurement of modification levels , Western blotting for protein-level detection, and immunoprecipitation for identifying proteins that interact with 2-hydroxyisobutyrylated H3K79. When combined with other techniques such as RNA-seq or ATAC-seq, these antibody-based approaches can reveal connections between H3K79 2-hydroxyisobutyrylation and transcriptional regulation, chromatin accessibility, or other epigenetic phenomena.

How should researchers design ChIP experiments using 2-hydroxyisobutyryl-HIST1H3A (K79) Antibody?

When designing ChIP experiments with 2-hydroxyisobutyryl-HIST1H3A (K79) Antibody, researchers should consider several critical factors to ensure optimal results. First, crosslinking conditions should be carefully optimized; standard formaldehyde crosslinking (1% for 10 minutes) works for most histone modifications, but pilot experiments with different crosslinking times may improve results for this specific modification. Sonication conditions should be adjusted to generate chromatin fragments of 200-500 bp for optimal immunoprecipitation efficiency. For antibody incubation, researchers should follow the manufacturer's recommended concentration or perform titration experiments (typically 2-5 μg per ChIP reaction) to determine optimal antibody amounts . Essential controls must include: (1) input DNA control, (2) IgG negative control from the same species as the antibody, (3) a positive control using an antibody against a well-characterized histone mark, and (4) ideally, a peptide competition control to confirm specificity. When analyzing the resulting data, researchers should normalize to input and compare enrichment patterns with known gene regulatory elements to interpret the biological significance of H3K79 2-hydroxyisobutyrylation distribution.

What are the optimal storage and handling conditions for 2-hydroxyisobutyryl-HIST1H3A (K79) Antibody?

Proper storage and handling of 2-hydroxyisobutyryl-HIST1H3A (K79) Antibody is crucial for maintaining its activity and specificity over time. Upon receipt, the antibody should be stored at -20°C or -80°C, with -80°C preferred for long-term storage . Repeated freeze-thaw cycles should be strictly avoided as they can lead to antibody degradation and loss of activity; aliquoting the antibody into single-use volumes upon receipt is strongly recommended. The antibody is typically supplied in a buffer containing 50% glycerol and 0.01M PBS (pH 7.4) with 0.03% Proclin 300 as a preservative , which helps maintain stability during storage. When working with the antibody, researchers should handle it on ice and return it to frozen storage promptly after use. For dilutions, it's best to use fresh buffer rather than repeatedly accessing the stock solution. For immunofluorescence applications, the recommended dilution range is 1:50-1:200 , but researchers should optimize this for their specific application and cell type. Following these handling practices will maximize antibody performance and extend its usable lifespan.

How does cellular metabolism influence H3K79 2-hydroxyisobutyrylation levels?

Cellular metabolism plays a crucial role in regulating H3K79 2-hydroxyisobutyrylation levels through multiple mechanisms. Research has demonstrated that unlike lysine acetylation, 2-hydroxyisobutyrylation shows a quantitative response to changes in glycolytic flux . This metabolic link suggests that energy metabolism directly impacts the epigenetic landscape through this modification. The 2-hydroxyisobutyryl group likely derives from 2-hydroxyisobutyrate, a metabolite connected to branched-chain amino acid metabolism and potentially glycolysis. During conditions of altered glucose metabolism, such as hypoxia or cancer-associated metabolic reprogramming, researchers can expect significant changes in H3K79 2-hydroxyisobutyrylation patterns. This creates an important research avenue for investigating how metabolic states influence gene expression through this specific epigenetic mark. Experimental approaches to study this connection include manipulating glycolytic activity through glucose availability, hypoxia, or glycolysis inhibitors, followed by measurement of global and locus-specific H3K79 2-hydroxyisobutyrylation levels using the antibody in Western blot, ChIP-seq, or immunofluorescence applications.

What enzymes regulate the addition and removal of 2-hydroxyisobutyrylation at H3K79?

The enzymatic regulation of 2-hydroxyisobutyrylation at H3K79 involves specific writers and erasers, though our understanding is still developing. Histone acetyltransferase Esa1p in budding yeast and its human homologue TIP60 have been demonstrated to catalyze the K hib reaction both in vitro and in vivo , suggesting these enzymes may function as 2-hydroxyisobutyryl transferases. For the removal of this modification, in vitro experiments have provided evidence that histone deacetylases 1-3 (HDAC 1-3), as well as yeast Rpd3p and Hos3p, can function as potential regulatory enzymes for lysine de-2-hydroxyisobutyrylation reactions on core histones . This dual functionality of certain HDACs as both deacetylases and de-2-hydroxyisobutyrylases suggests interesting crosstalk between these two modification pathways. To study the enzyme-substrate relationship specifically for H3K79, researchers can employ approaches such as in vitro enzymatic assays with recombinant enzymes and H3K79-containing peptides, followed by mass spectrometry or antibody-based detection. In cellular contexts, enzyme knockdown/knockout experiments combined with ChIP-seq using the 2-hydroxyisobutyryl-HIST1H3A (K79) Antibody can reveal enzyme-dependent changes in modification distribution.

What sequence motifs are associated with 2-hydroxyisobutyrylation sites, and how do they compare with H3K79?

Comprehensive analysis of lysine 2-hydroxyisobutyrylation sites has revealed distinct sequence motifs surrounding modified lysines. According to proteome-wide studies, there is a strong bias for negatively charged amino acids, particularly aspartic acid (D) and glutamic acid (E), around the modified lysine residues . The three most enriched motifs identified were [EK hib], [D XXK hib], and [K hibE], indicating a preference for acidic residues adjacent to modification sites . Besides D and E, lysine (K) at -9 position, arginine (R) at +8 position, and valine (V) at -1 and +2 positions were over-represented around modified lysine sites . In contrast, lysine (K), proline (P), arginine (R), and serine (S) at positions -4 to +4 were under-represented around 2-hydroxyisobutyrylated lysines . When examining the H3K79 site specifically, researchers should consider how its local sequence context compares to these general motifs. This sequence specificity likely influences which enzymes can modify particular sites and may help explain the biological specificity of 2-hydroxyisobutyrylation across the proteome. Sequence logo analysis and comparison of H3K79 with other 2-hydroxyisobutyrylation sites can provide insights into the unique features of this specific modification site.

What are common challenges when using 2-hydroxyisobutyryl-HIST1H3A (K79) Antibody and how can they be addressed?

Researchers working with 2-hydroxyisobutyryl-HIST1H3A (K79) Antibody may encounter several common challenges that require systematic troubleshooting. One frequent issue is weak or non-specific signal in immunoblotting or immunofluorescence applications. This can be addressed by: (1) optimizing antibody concentration through careful titration experiments, starting with the recommended dilution of 1:50-1:200 for immunofluorescence , (2) extending primary antibody incubation time or temperature (typically overnight at 4°C provides better results than shorter incubations), (3) using more sensitive detection methods such as enhanced chemiluminescence for Western blots or signal amplification systems for immunofluorescence, and (4) optimizing blocking conditions to reduce background. Another common challenge is poor immunoprecipitation efficiency in ChIP experiments, which may be improved by increasing chromatin amount, optimizing sonication conditions, or adjusting wash stringency. Cross-reactivity with other histone modifications presents another potential issue, which can be addressed through careful validation using peptide competition assays with modified and unmodified peptides. Finally, batch-to-batch variation in antibody performance can be mitigated by maintaining consistent validation protocols and keeping reference samples to compare antibody performance across experiments.

How can researchers validate the specificity of 2-hydroxyisobutyryl-HIST1H3A (K79) Antibody?

Validating the specificity of 2-hydroxyisobutyryl-HIST1H3A (K79) Antibody is crucial for generating reliable experimental data. A comprehensive validation approach should include multiple complementary methods. Peptide competition assays represent a gold standard validation technique, where pre-incubating the antibody with excess 2-hydroxyisobutyrylated H3K79 peptide should abolish signal, while pre-incubation with unmodified peptide or peptides containing other modifications at K79 should not affect antibody binding. Dot blot assays with modified and unmodified peptides can provide quantitative data on antibody specificity and sensitivity. Western blot analysis comparing wild-type samples with samples where H3K79 is mutated (K79R or K79A) can confirm antibody specificity in a cellular context. For in situ applications, immunofluorescence staining patterns should be compared between control cells and cells treated with HDAC inhibitors or metabolic modulators known to affect 2-hydroxyisobutyrylation levels. Mass spectrometry analysis of immunoprecipitated material can provide additional confirmation of antibody specificity by identifying the exact modification on the target residue. Implementing these validation approaches ensures that experimental observations truly reflect H3K79 2-hydroxyisobutyrylation rather than other modifications or non-specific binding.

What are the critical sample preparation considerations for studying H3K79 2-hydroxyisobutyrylation?

Sample preparation represents a critical determinant of success when studying H3K79 2-hydroxyisobutyrylation with antibody-based methods. One key consideration is the preservation of the modification during extraction and processing. Researchers should include deacetylase inhibitors (such as sodium butyrate, trichostatin A, or nicotinamide) in all buffers to prevent removal of the modification during sample handling. Additionally, protease inhibitors must be included to prevent degradation of histones. For nuclear extraction and histone isolation, gentle methods that preserve native modifications should be employed, such as acid extraction protocols specifically designed for histones. When preparing samples for immunofluorescence, fixation conditions should be optimized; paraformaldehyde fixation (typically 4% for 10-15 minutes) is generally suitable, but overfixation may mask epitopes. For chromatin preparation in ChIP experiments, crosslinking conditions and sonication parameters should be carefully optimized to ensure efficient fragmentation while preserving the modification. All samples should be processed quickly at cold temperatures (4°C) whenever possible to minimize enzymatic removal of modifications. Finally, researchers should consider the metabolic state of cells during harvest, as glycolytic activity can influence 2-hydroxyisobutyrylation levels , potentially requiring standardized cell culture and harvest conditions for reproducible results.

How should ChIP-seq data for H3K79 2-hydroxyisobutyrylation be analyzed and interpreted?

Analysis and interpretation of ChIP-seq data for H3K79 2-hydroxyisobutyrylation requires several specialized considerations beyond standard ChIP-seq workflows. After quality control and alignment of sequencing reads to the reference genome, peak calling should be performed using algorithms suitable for histone modifications, such as MACS2 with broad peak settings, as histone modifications often cover extended genomic regions. Normalization is critical; researchers should normalize to input control and consider sequence bias correction methods. For comparative analysis across conditions, quantile normalization or spike-in normalization approaches may be necessary to account for global changes in modification levels. Researchers should examine the distribution of H3K79 2-hydroxyisobutyrylation relative to genomic features (promoters, gene bodies, enhancers) and integrate this data with other epigenomic datasets (e.g., other histone modifications, transcription factor binding, chromatin accessibility) to understand the functional context. Correlation with transcriptomic data can reveal relationships between H3K79 2-hydroxyisobutyrylation and gene expression. Motif enrichment analysis within peak regions may identify transcription factors associated with this modification. Given the metabolic connection of 2-hydroxyisobutyrylation, researchers should consider analyzing the data in the context of metabolic pathways or under conditions of altered metabolism. Tools like Genomic Regions Enrichment of Annotations Tool (GREAT) can help identify biological processes associated with H3K79 2-hydroxyisobutyrylated regions.

How does H3K79 2-hydroxyisobutyrylation compare to other histone modifications in functional genomics studies?

Comparing H3K79 2-hydroxyisobutyrylation with other histone modifications can provide valuable insights into its unique functional roles in chromatin regulation. Unlike many histone modifications that occur primarily on N-terminal tails, H3K79 is located in the globular domain of histone H3, potentially affecting nucleosome structure and stability in distinct ways. The stoichiometry of H3K79 2-hydroxyisobutyrylation in synchronized G2/M HeLa cells is comparable to or even higher than many established histone acetylation marks, suggesting significant biological importance . In genomic distribution studies, researchers should compare H3K79 2-hydroxyisobutyrylation patterns with other well-characterized modifications such as H3K4me3 (associated with active promoters), H3K36me3 (associated with gene bodies of transcribed genes), and H3K27ac (associated with active enhancers). This comparative approach can reveal whether H3K79 2-hydroxyisobutyrylation marks distinct regulatory elements or overlaps with known functional domains. The dynamic changes of H3K79 2-hydroxyisobutyrylation during cell cycle progression or in response to metabolic fluctuations may differ from other modifications, providing clues to its specific regulatory functions. Integrated analysis of multiple histone modifications, including H3K79 2-hydroxyisobutyrylation, can help construct comprehensive epigenetic signatures associated with specific cellular states or developmental processes.

What bioinformatic resources and tools are most valuable for 2-hydroxyisobutyrylation research?

Researchers investigating H3K79 2-hydroxyisobutyrylation can leverage several specialized bioinformatic resources and tools to enhance their analyses. For proteome-wide studies of 2-hydroxyisobutyrylation sites, databases like PTMfunc, PLMD (Protein Lysine Modification Database), and PTMCode provide valuable resources for comparison with published datasets. The PRIDE repository (dataset identifier PXD005986) contains mass spectrometry data on 2-hydroxyisobutyrylation sites that researchers can access for comparative analyses . For motif analysis around modification sites, tools like Motif-X, MoMo, and pLogo can identify sequence patterns that may relate to enzyme specificity. NetSurfP can be used for secondary structure analysis of 2-hydroxyisobutyrylation sites, as previous research has shown that these sites tend to have lower surface accessibility compared to unmodified lysines . For functional enrichment analyses, tools like DAVID, GREAT, Metascape, or g:Profiler can identify biological processes associated with 2-hydroxyisobutyrylated proteins or genomic regions. For integrative analysis of ChIP-seq data with other epigenomic or transcriptomic datasets, platforms like Galaxy, Cistrome, or EpiGenome Browser provide user-friendly interfaces. Computational prediction of 2-hydroxyisobutyrylation sites based on sequence and structural features can be performed using machine learning tools like DeepKhib or iHyd-PseAAC. These resources collectively enhance the ability of researchers to extract biological insights from 2-hydroxyisobutyrylation data.

What are the emerging research areas related to H3K79 2-hydroxyisobutyrylation?

Several exciting research frontiers are emerging in the field of H3K79 2-hydroxyisobutyrylation. One promising direction involves investigating the crosstalk between H3K79 2-hydroxyisobutyrylation and other histone modifications, particularly those occurring in proximity on the nucleosome surface that may functionally interact. The development of new technologies for site-specific introduction of 2-hydroxyisobutyrylated lysine into recombinant histones, such as the amber suppression-mediated strategy , opens avenues for precise mechanistic studies of how this modification affects chromatin structure and protein-DNA interactions. The metabolic regulation of 2-hydroxyisobutyrylation and its potential role in communicating cellular metabolic states to chromatin represents another frontier, particularly in contexts like cancer metabolism, stem cell differentiation, and aging. The identification and characterization of specific "reader" proteins that recognize and bind to 2-hydroxyisobutyrylated H3K79 would significantly advance our understanding of how this modification exerts its biological effects. Single-cell epigenomic technologies applied to 2-hydroxyisobutyrylation could reveal cell-to-cell heterogeneity in modification patterns and their correlation with cellular states. Finally, translational research examining the role of H3K79 2-hydroxyisobutyrylation in disease states and its potential as a biomarker or therapeutic target represents an important emerging area with clinical implications.

How might therapeutic targeting of H3K79 2-hydroxyisobutyrylation be developed?

The therapeutic targeting of H3K79 2-hydroxyisobutyrylation represents a promising frontier in epigenetic medicine, though currently at an early conceptual stage. Development strategies could focus on several approaches. Small molecule inhibitors or activators of the enzymes regulating 2-hydroxyisobutyrylation, such as the identified writer enzyme TIP60 or eraser enzymes HDAC1-3 , could modulate global or locus-specific 2-hydroxyisobutyrylation levels. Metabolic approaches targeting the cellular pathways that generate 2-hydroxyisobutyrate or regulate glycolytic flux could indirectly modulate 2-hydroxyisobutyrylation levels, potentially with fewer off-target effects than direct enzyme inhibition. Peptide or small molecule compounds designed to interfere with the binding of "reader" proteins to 2-hydroxyisobutyrylated H3K79 could disrupt specific downstream effects of the modification. Combined approaches targeting multiple epigenetic modifications simultaneously, including H3K79 2-hydroxyisobutyrylation, might overcome the compensatory mechanisms that often limit single-target epigenetic therapies. For developing such therapeutics, high-throughput screening assays using the 2-hydroxyisobutyryl-HIST1H3A (K79) Antibody could identify compounds that modulate this modification. Disease-relevant model systems where H3K79 2-hydroxyisobutyrylation plays a documented role should be prioritized for initial therapeutic testing, with careful assessment of specificity, efficacy, and potential side effects through comprehensive epigenomic profiling.

What technological advances would accelerate research on H3K79 2-hydroxyisobutyrylation?

Several technological innovations would significantly accelerate research on H3K79 2-hydroxyisobutyrylation. Development of even more specific antibodies with higher affinity and reduced lot-to-lot variation would enhance detection sensitivity and reproducibility across laboratories. Antibodies capable of distinguishing between 2-hydroxyisobutyrylation at specific lysine residues within the same protein would be particularly valuable. CRISPR-based epigenome editing tools specifically targeting 2-hydroxyisobutyrylation machinery to defined genomic loci would enable causal studies of this modification's role in gene regulation. Mass spectrometry methods with improved sensitivity for detecting and quantifying 2-hydroxyisobutyrylated peptides, including targeted approaches for H3K79, would enhance our ability to study stoichiometry and dynamics of this modification. Live-cell imaging tools for visualizing 2-hydroxyisobutyrylation dynamics in real-time, perhaps using engineered reader domains coupled to fluorescent proteins, would reveal temporal aspects of regulation currently inaccessible. Single-molecule techniques for analyzing how 2-hydroxyisobutyrylation affects nucleosome dynamics and stability would provide mechanistic insights at unprecedented resolution. Single-cell ChIP-seq or CUT&Tag methods adapted for 2-hydroxyisobutyrylation would reveal cell-to-cell heterogeneity and rare cell populations with distinct epigenetic states. Finally, improved computational methods for integrating 2-hydroxyisobutyrylation data with other omics datasets would enhance our ability to extract biological meaning from increasingly complex multi-dimensional datasets.