2-hydroxyisobutyryl-HIST1H2AG (K9) Antibody

What is 2-hydroxyisobutyryl-HIST1H2AG (K9) Antibody and what is its role in epigenetic research?

2-Hydroxyisobutyryl-HIST1H2AG (K9) Antibody is a polyclonal antibody designed to specifically recognize the 2-hydroxyisobutyrylation modification at lysine 9 of the histone H2A type 1 protein (HIST1H2AG). This post-translational modification plays a crucial role in epigenetic regulation.

The antibody serves as a vital tool for investigating histone modifications that influence chromatin structure and gene expression. 2-Hydroxyisobutyrylation (Khib) specifically represents a novel class of histone marks identified at numerous lysine sites in human and mouse histones, including many unique sites that had not been previously associated with other modifications .

For optimal antibody performance, follow these research-validated storage guidelines:

• Short-term storage (up to 2 weeks): Maintain refrigerated at 2-8°C

• Long-term storage: Store at -20°C or -80°C in small aliquots

• Avoid repeated freeze-thaw cycles as this can compromise antibody activity

• The antibody is typically provided in a buffer containing preservatives (0.03% Proclin 300) and stabilizers (50% Glycerol) in PBS (pH 7.4)

• Working dilutions should be prepared fresh before use in experiments

How can I validate the specificity of 2-hydroxyisobutyryl-HIST1H2AG (K9) Antibody in my experimental system?

Validating antibody specificity is critical for reliable experimental results. Implement the following methodological approach:

• Peptide competition assay: Pre-incubate the antibody with increasing concentrations of the immunizing peptide (containing 2-hydroxyisobutyryl-K9) before application in your experiment. Signal reduction confirms specificity.

• Cross-reactivity testing: Test against samples containing related histone modifications such as acetylation or other 2-hydroxyisobutyrylation sites. Research shows distinct recognition patterns between different histone PTMs despite chemical similarities .

• Knockout/knockdown validation: Use CRISPR/Cas9 to eliminate the target protein or enzymes responsible for the modification (such as P300, which can catalyze 2-hydroxyisobutyrylation ).

• Mass spectrometry correlation: Confirm antibody-detected signals with mass spectrometry analysis to independently verify the presence of the modification at the expected molecular weight.

• Multiple antibody validation: Compare results using antibodies from different sources or clones that recognize the same epitope.

What controls should I include when using this antibody for ChIP experiments?

For robust ChIP experiments with 2-hydroxyisobutyryl-HIST1H2AG (K9) Antibody, include these essential controls:

• Input control: Reserve 5-10% of chromatin before immunoprecipitation to normalize for DNA abundance.

• No-antibody control: Perform IP procedure without primary antibody to identify non-specific binding.

• IgG control: Use matched rabbit IgG at the same concentration as the specific antibody .

• Positive control regions: Include genomic regions known to be enriched for H2A K9 2-hydroxyisobutyrylation. Based on research, active gene promoters are likely to show this modification .

• Negative control regions: Include heterochromatin regions like γ-satellite, which typically show low levels of active histone marks. Research shows that repressed genes like Hist1h2aa have histone H3 K9 acetylation levels similar to γ-satellite regions .

• Technical replicates: Perform at least three technical replicates to ensure reproducibility.

How does 2-hydroxyisobutyrylation at HIST1H2AG K9 differ functionally from other histone modifications at the same residue?

Lysine 9 of histone H2A can undergo multiple modifications including acetylation, methylation, and 2-hydroxyisobutyrylation, each with distinct functional outcomes:

Research indicates that 2-hydroxyisobutyrylation serves as a distinct epigenetic mark with potential roles in transcriptional regulation that may both overlap with and differ from acetylation. Current evidence suggests that 2-hydroxyisobutyrylation marks are enriched at active gene promoters, similar to acetylation, but may regulate distinct sets of genes under specific metabolic conditions .

The functional distinction is particularly important when investigating metabolic regulation of gene expression, as 2-hydroxyisobutyrylation connects cellular metabolism to epigenetic regulation in ways that other modifications may not.

What is known about the role of histone H2A 2-hydroxyisobutyrylation in cell-cycle regulation and cellular stress responses?

Research on histone H2A 2-hydroxyisobutyrylation in cell cycle and stress responses remains emerging, but related findings suggest:

• Cell cycle regulation: While specific data on H2A K9 2-hydroxyisobutyrylation is limited, studies on related histone H2A variants show distinct expression patterns throughout the cell cycle. Replication-dependent histone H2A genes (including HIST1H2AG) typically show increased expression during S-phase . This suggests that 2-hydroxyisobutyrylation levels may fluctuate in a cell-cycle dependent manner.

• Metabolic stress connection: 2-Hydroxyisobutyrylation is chemically related to β-hydroxybutyrylation (Kbhb), which increases during metabolic stress conditions like starvation or ketogenic diet . This suggests 2-hydroxyisobutyrylation may similarly respond to metabolic status, creating a potential direct link between cellular metabolism and chromatin regulation.

• Differentiation and development: Histone H2A variants show differential expression during cellular differentiation, suggesting that their post-translational modifications, including 2-hydroxyisobutyrylation, may play regulatory roles in development .

The investigation of these relationships requires careful experimental design with synchronized cell populations and metabolic manipulation coupled with ChIP-seq or mass spectrometry analysis.

How can I investigate the interplay between 2-hydroxyisobutyrylation and other histone modifications in promoter regions?

To interrogate the functional interplay between histone modifications:

• Sequential ChIP (ReChIP): Perform ChIP with 2-hydroxyisobutyryl-HIST1H2AG (K9) antibody followed by a second ChIP with antibodies against other modifications (e.g., H3K4me3, H3K27ac) to identify genomic regions with co-occurrence.

• Integrated genomics approach: Combine ChIP-seq data for multiple histone modifications with transcriptome analysis to correlate modification patterns with gene expression levels. Research on histone H3 K9 acetylation demonstrates correlation with gene expression levels of histone genes themselves .

• Enzyme inhibition studies: Use specific inhibitors of histone-modifying enzymes to disrupt one modification and observe effects on others. For example, HDAC inhibitors might alter the balance between acetylation and 2-hydroxyisobutyrylation.

• Mass spectrometry analysis: Employ quantitative mass spectrometry to determine the stoichiometry of different modifications on the same histone molecule.

• Single-molecule imaging: Use super-resolution microscopy with differentially labeled antibodies to visualize the spatial relationship between different modifications in the nucleus.

How can I optimize Western blot protocols when working with 2-hydroxyisobutyryl-HIST1H2AG (K9) Antibody?

For optimal Western blot results with this antibody:

• Histone extraction: Use specialized acid extraction protocols for histones to ensure efficient isolation of nuclear proteins:

  • Extract histones with 0.2N HCl or 0.4N H2SO4

  • Precipitate with trichloroacetic acid (TCA)

  • Wash with acetone containing 0.1% HCl

  • Air-dry and resuspend in water

• Gel selection: Use high-percentage (15-18%) SDS-PAGE gels or specialized Triton-Acid-Urea gels to achieve better separation of histone proteins, which have low molecular weights (~14-17 kDa) .

• Transfer optimization: Use PVDF membranes and semi-dry transfer systems with specialized buffers (containing 0.05% SDS) to ensure efficient transfer of small histone proteins.

• Blocking optimization: Use 5% BSA rather than milk for blocking to prevent non-specific interactions.

• Antibody concentration: Start with a 1:1000 dilution for Western blot and optimize as needed. Some protocols suggest ranges from 1:500-1:5000 .

• Signal detection: Use enhanced chemiluminescence (ECL) detection systems with increased sensitivity for optimal visualization of specific bands at approximately 14 kDa.

• Controls: Include both positive control samples (e.g., cells known to have high levels of the modification) and loading controls (e.g., total H2A or total histone H3) .

What are the common pitfalls when using this antibody for immunofluorescence and how can they be addressed?

When using 2-hydroxyisobutyryl-HIST1H2AG (K9) antibody for immunofluorescence, researchers may encounter these challenges:

• High background signal:

  • Solution: Optimize blocking (try 5% BSA, 3% normal serum, or commercial blocking reagents)

  • Increase washing duration and frequency (use 0.1% Triton X-100 in PBS)

  • Titrate antibody concentration (start with 1:50-1:100 dilution)

• Weak specific signal:

  • Solution: Enhance antigen retrieval (try heat-mediated retrieval with citrate buffer pH 6.0)

  • Increase antibody incubation time (overnight at 4°C)

  • Use signal amplification systems (tyramide signal amplification or secondary antibody with higher fluorophore conjugation)

• Nuclear penetration issues:

  • Solution: Optimize fixation and permeabilization (4% paraformaldehyde followed by 0.5% Triton X-100)

  • Consider alternative protocols using methanol/acetone fixation

• Specificity concerns:

  • Solution: Pre-adsorb antibody with peptides containing unmodified K9 to remove cross-reactive antibodies

  • Include appropriate controls (cells with known high/low levels of the modification)

  • Validate observations with orthogonal techniques such as Western blot

According to available data, optimal dilution ranges for immunofluorescence applications are approximately 1:50-1:200 .

How does the expression and modification pattern of HIST1H2AG compare to other histone H2A variants?

Research on histone H2A variants reveals differential expression patterns that impact interpretation of modification data:

• Expression levels: Among 20 histone H2A genes studied in mouse, the replication-independent H2AFZ shows the highest expression level, while HIST1H2AA (replication-dependent) shows the lowest. Among replication-dependent H2A genes, HIST3H2A demonstrates the highest expression .

• Cell-cycle dependence: Replication-dependent H2A genes (including HIST1H2AG) show peak expression during S-phase and decreased expression at the end of S-phase, while replication-independent variants like H2AFZ show more stable expression .

• Promoter regulation: Histone H3 K9 acetylation levels in promoter regions correlate with expression levels of histone genes. The promoters of highly expressed variants like H2AFZ and HIST3H2A show significantly higher H3 K9 acetylation compared to poorly expressed variants like HIST1H2AA .

When interpreting 2-hydroxyisobutyrylation data, consider these expression differences, as modification levels may correlate with underlying gene expression patterns.

What are the differences between 2-hydroxyisobutyrylation at K9 versus other lysine sites on HIST1H2AG?

Multiple lysine residues on HIST1H2AG can undergo 2-hydroxyisobutyrylation, with distinct functional implications:

Research indicates that 2-hydroxyisobutyrylation can occur at multiple lysine residues within histones, with K9 being one of the most extensively studied. Different lysine modifications likely serve distinct functions in regulating chromatin structure and gene expression.

The comprehensive analysis of modification patterns across multiple lysine residues requires mass spectrometry approaches or the use of modification-specific antibodies for each site. According to published data on histone modifications, different lysine residues show varying susceptibility to different types of acylation, suggesting site-specific regulation by writer and eraser enzymes .

How does the dynamics of 2-hydroxyisobutyrylation compare with other acyl modifications in response to metabolic changes?

Research on histone acylations reveals differential responses to metabolic conditions:

• Metabolic sensitivity:

  • 2-Hydroxyisobutyrylation (Khib) is influenced by cellular levels of 2-hydroxyisobutyrate (HIB)

  • β-Hydroxybutyrylation (Kbhb) increases during starvation or ketogenic diet when β-hydroxybutyrate (BHB) levels rise

  • Acetylation (Kac) correlates with acetyl-CoA levels, a central metabolic intermediate

• Enzymatic regulation:

  • P300 can catalyze multiple acylations including Khib, Kbhb, and Kac

  • Different HDAC/SIRT family members show specificity for different acyl groups

  • SIRT3 preferentially removes Kbhb while HDAC1-3 target Khib

• Functional outcomes:

  • Kac is often associated with chronic inflammatory conditions

  • Kbhb upregulates starvation-responsive metabolic pathway genes

  • Khib affects genes involved in meiosis and post-meiotic development

These differences suggest that each acyl modification serves as a distinct metabolic sensor, translating specific cellular metabolic states into epigenetic regulation. When designing experiments to study 2-hydroxyisobutyrylation dynamics, researchers should consider manipulating cellular metabolism (e.g., through nutrient restriction or supplementation) while monitoring multiple acyl modifications simultaneously.