2-hydroxyisobutyryl-HIST1H1C (K116) Antibody

What is 2-hydroxyisobutyryl-HIST1H1C (K116) Antibody and what does it detect?

The 2-hydroxyisobutyryl-HIST1H1C (K116) antibody is a polyclonal antibody raised in rabbits against a peptide sequence surrounding the 2-hydroxyisobutyrylated lysine 116 residue in human Histone H1.2 (also known as H1c or HIST1H1C) . This antibody specifically recognizes a post-translational modification (PTM) at a specific position within the C-terminal domain (CTD) of the histone H1.2 protein. The 2-hydroxyisobutyrylation represents one of many PTMs that can occur on histone proteins and is believed to play important roles in chromatin regulation and gene expression. Unlike general H1.2 antibodies, this reagent specifically detects only the subset of H1.2 proteins that carry this particular modification at lysine 116.

How does HIST1H1C compare to other H1 histone variants?

HIST1H1C (H1.2) is one of several H1 histone variants expressed in human cells. These variants share high sequence homology, with approximately 74-87% sequence identity between the most common somatic variants . The main differences between H1 variants are primarily located in their amino and carboxy terminal tails . HIST1H1C plays important roles in chromatin structure and gene regulation, and it may have distinct functions compared to other variants. For example, overexpression experiments have shown that different H1 variants can yield distinct phenotypes, with some affecting cell cycle progression while others influence transcription levels . The CTD of histone H1 variants determines their binding affinity for nucleosomes, with differences in the CTD length affecting residence times of variants on nucleosomes .

What applications is this antibody suitable for?

The 2-hydroxyisobutyryl-HIST1H1C (K116) antibody has been validated for several applications in research settings:

This antibody has been specifically designed for these applications in human samples . Its high specificity for the 2-hydroxyisobutyryl modification at K116 makes it valuable for studying this particular PTM in various experimental contexts.

How can I validate the specificity of this antibody in my experimental system?

Validating antibody specificity is crucial, particularly for histone PTM antibodies where cross-reactivity with similar modifications is a concern. For the 2-hydroxyisobutyryl-HIST1H1C (K116) antibody, implement the following validation steps:

• Peptide competition assay: Pre-incubate the antibody with excess synthetic peptides containing the 2-hydroxyisobutyryl-K116 modification versus unmodified peptides, then perform Western blot analysis. Signal reduction with the modified peptide indicates specificity.

• Knockout/knockdown controls: Compare signal between wild-type cells and cells where HIST1H1C has been knocked down or out.

• Dot blot analysis: Test the antibody against a panel of synthetic peptides with various modifications at and around K116 to assess cross-reactivity.

• Mass spectrometry correlation: Validate antibody results by performing immunoprecipitation followed by mass spectrometry to confirm the presence of the 2-hydroxyisobutyryl modification.

What are the optimal sample preparation methods for chromatin immunoprecipitation (ChIP) with this antibody?

For optimal ChIP results using the 2-hydroxyisobutyryl-HIST1H1C (K116) antibody:

• Crosslinking: Use 1% formaldehyde for 10 minutes at room temperature. The high lysine content in histone H1 CTD regions requires careful crosslinking optimization as excessive crosslinking may mask the epitope .

• Chromatin fragmentation: Sonicate to achieve fragment sizes of 200-500bp. H1 histones are more loosely associated with chromatin than core histones, so gentler sonication conditions may be required.

• Immunoprecipitation: Use 2-5μg of antibody per ChIP reaction with 25-50μg of chromatin input.

• Washing conditions: Include high-salt washes to reduce non-specific binding.

• Controls: Include IgG negative control, input control, and positive controls such as antibodies against known histone marks.

Remember that histone H1 is known to have dynamic interactions with chromatin, with residence times in the range of minutes, which is different from the more stable core histones . This dynamic nature may affect ChIP efficiency and should be considered when interpreting results.

How does 2-hydroxyisobutyrylation at K116 compare functionally to other modifications on histone H1?

The 2-hydroxyisobutyrylation at K116 in HIST1H1C represents one of many PTMs that can occur on histone H1 variants. The functional significance should be considered in context with other known H1 modifications:

• Phosphorylation of H1 has been extensively studied and has dual functions in chromatin condensation and decondensation depending on site and cell cycle context . For example, phosphorylation can disrupt interaction between H1 and heterochromatin protein 1α, leading to chromatin decondensation .

• The location of K116 in the CTD of H1.2 is significant because the CTD is critical for chromatin binding. The CTD contains many lysine residues that contribute to its net positive charge, which enables DNA backbone charge neutralization and formation of higher-order chromatin structures .

• While phosphorylation affects H1 residence time on chromatin, 2-hydroxyisobutyrylation likely alters the charge properties of the lysine residue, potentially affecting the CTD's ability to interact with and condense DNA.

• Unlike acetylation (which neutralizes the positive charge of lysine) or methylation (which preserves the charge), 2-hydroxyisobutyrylation adds a bulky group that may have unique functional consequences for chromatin structure.

When studying 2-hydroxyisobutyryl-HIST1H1C (K116), consider that H1 variants often contain multiple simultaneous PTMs, and the biological effects may result from combinations of modifications rather than a single PTM .

What are common challenges when working with histone H1 antibodies?

Working with histone H1 antibodies presents several technical challenges:

• High sequence homology between variants: H1 variants share 74-87% sequence identity, making generation of variant-specific antibodies challenging . The 2-hydroxyisobutyryl-HIST1H1C (K116) antibody targets both a specific variant and modification, reducing but not eliminating cross-reactivity concerns.

• Multiple simultaneous PTMs: Histone H1 variants frequently contain multiple PTMs simultaneously, which can affect antibody recognition . The terminal tails of histone H1 variants are among the most abundantly modified sequences in the cell, with modifications that can influence antibody binding .

• Dynamic nature of H1: The relatively short residence time of H1 histones on chromatin (minutes versus hours for core histones) can affect experimental outcomes, particularly in ChIP experiments .

• Sample preparation: Traditional sample preparation methods optimized for core histones may not be optimal for H1 histones due to differences in biochemical properties and chromatin association.

To address these challenges, include appropriate controls, validate the antibody thoroughly in your experimental system, and consider complementing antibody-based approaches with mass spectrometry when possible.

How should I prepare samples for Western blot analysis using this antibody?

For optimal results in Western blot analysis with the 2-hydroxyisobutyryl-HIST1H1C (K116) antibody:

• Extraction: Use specialized histone extraction protocols with high salt (0.4M NaCl) to effectively extract histone H1 proteins from chromatin.

• Sample preparation: Add histone deacetylase inhibitors (like sodium butyrate) and phosphatase inhibitors to preserve PTMs during extraction.

• Gel electrophoresis: Use 15% SDS-PAGE gels for optimal resolution of histone proteins.

• Transfer conditions: Use PVDF membranes and wet transfer methods for efficient transfer of the highly basic histone proteins.

• Blocking: Use 5% BSA instead of milk to reduce background, as milk contains proteins that can cross-react with some histone antibodies.

• Antibody incubation: Dilute the antibody properly in 1% BSA and incubate overnight at 4°C for optimal results.

• Positive controls: Include samples from cells with known high levels of the modification, if available.

These recommendations account for the unique properties of histone H1 proteins and their PTMs. The high lysine content of histone H1, particularly in the CTD, can affect extraction and detection efficiency .

How might 2-hydroxyisobutyrylation of HIST1H1C (K116) change during cell cycle progression?

Based on our understanding of histone H1 PTMs during cell cycle progression:

• Histone H1 phosphorylation is known to progressively increase as cells progress from G1 to mitosis, with different phases of phosphorylation having distinct functions . While less is known specifically about 2-hydroxyisobutyrylation dynamics, it may follow similar cell cycle-dependent patterns.

• During interphase, H1 modifications often regulate transcriptional accessibility. The 2-hydroxyisobutyrylation of K116 might be involved in regulating access of transcription factors or chromatin remodeling complexes to specific genomic regions .

• During mitosis, histone H1 undergoes hyperphosphorylation, leading to chromatin condensation . The relationship between 2-hydroxyisobutyrylation and phosphorylation during mitosis should be investigated to understand potential crosstalk between these modifications.

• If 2-hydroxyisobutyrylation follows patterns similar to other histone acylations, it may be linked to metabolic state changes throughout the cell cycle.

When studying cell cycle-dependent changes, synchronize cells using appropriate methods, collect samples at defined time points, and assess both the modification level and total histone H1.2 abundance to distinguish between modification changes and protein level changes.

What genomic regions might be enriched for 2-hydroxyisobutyryl-HIST1H1C (K116) in ChIP-seq experiments?

When analyzing ChIP-seq data for 2-hydroxyisobutyryl-HIST1H1C (K116), researchers might expect:

• Different distribution patterns compared to core histone modifications: Histone H1 generally associates with linker DNA rather than nucleosomal DNA, resulting in distinct genomic distribution patterns.

• Potential association with specific chromatin states: Other histone acylations are often associated with active transcription, so 2-hydroxyisobutyryl-HIST1H1C may be enriched at actively transcribed regions.

• Possible correlation with DNA methylation: H1 histones are known to interact with DNA-modifying enzymes like DNMT1 and DNMT3B, suggesting potential co-localization with specific DNA methylation patterns .

• Cell type-specific patterns: Research has shown that H1 variants can have cell type-specific functions and genomic distributions.

When analyzing ChIP-seq data, consider using specialized peak calling algorithms optimized for histone modifications rather than transcription factors, and compare distributions with other histone marks and chromatin features to gain functional insights.

How can 2-hydroxyisobutyryl-HIST1H1C (K116) antibody be combined with other techniques for comprehensive epigenetic analysis?

For comprehensive epigenetic analysis, integrate this antibody with complementary techniques:

The integration of multiple techniques addresses the limitations of individual methods and provides more comprehensive insights into the biological functions of 2-hydroxyisobutyryl-HIST1H1C (K116).