2-hydroxyisobutyryl-HIST1H1C (K25) Antibody

What is HIST1H1C and what role does the K25 residue play in its function?

HIST1H1C (also known as Histone H1.2) is an important variant of the linker histone H1 family. It binds to linker DNA between nucleosomes, forming the macromolecular structure known as the chromatin fiber. Histones H1 are necessary for the condensation of nucleosome chains into higher-order structured fibers . The K25 (lysine 25) residue is located in the N-terminal domain of HIST1H1C and represents a critical site for post-translational modifications that can alter chromatin structure and regulate gene expression. This specific lysine residue, when modified by 2-hydroxyisobutyrylation, may influence chromatin accessibility and subsequent gene regulation in epigenetic mechanisms .

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

2-hydroxyisobutyrylation is a relatively newly identified post-translational modification of histones that involves the addition of a 2-hydroxyisobutyryl group to lysine residues. Unlike better-studied modifications such as acetylation or methylation, 2-hydroxyisobutyrylation creates a unique chemical environment that can distinctly alter protein-protein interactions and chromatin structure. This modification has been found to be particularly abundant during specific cellular processes and may represent a unique regulatory mechanism distinct from other histone marks. Research indicates that 2-hydroxyisobutyrylation at specific lysine residues like K25 on HIST1H1C may play specialized roles in chromatin dynamics and gene expression regulation in various biological contexts .

What are the recommended applications for the 2-hydroxyisobutyryl-HIST1H1C (K25) antibody?

The 2-hydroxyisobutyryl-HIST1H1C (K25) antibody is specifically designed for detecting human HIST1H1C that has been modified with a 2-hydroxyisobutyryl group at the K25 position. According to product specifications, this antibody is recommended for several experimental applications:

The antibody has been affinity-purified against the specific modified peptide sequence around the 2-hydroxyisobutyryl-K25 site of human HIST1H1C, ensuring specificity for this particular modification . While not explicitly indicated in all product information, related antibodies to modified HIST1H1C may also be applicable for Western blotting (WB) and chromatin immunoprecipitation (ChIP) techniques as seen with similar histone modification antibodies .

How should samples be prepared for optimal detection of 2-hydroxyisobutyryl-HIST1H1C (K25)?

For optimal detection of 2-hydroxyisobutyryl-HIST1H1C (K25), sample preparation should consider the preservation of nuclear proteins and their modifications. Based on experimental protocols with similar histone modification antibodies:

• For immunofluorescence and immunocytochemistry:

  • Cells should be fixed with paraformaldehyde (typically 4%) to preserve protein structure and modifications

  • Permeabilization with 0.25% Triton X-100/PBS is recommended to allow antibody access to nuclear proteins

  • Consider antigen retrieval methods if working with tissue sections

• For cellular fractionation and protein extraction:

  • Nuclear/cytoplasmic fractionation assays can be valuable, as HIST1H1C is primarily localized in the nucleus

  • Acid extraction methods (using sulfuric acid or hydrochloric acid) are often preferred for histone isolation

  • Deacetylase inhibitors (such as sodium butyrate) and protease inhibitors should be included in buffers to preserve modifications

• Storage considerations:

  • Upon receipt, antibody should be stored at -20°C or -80°C

  • Avoid repeated freeze-thaw cycles to maintain antibody activity

What roles does HIST1H1C play in autophagy and how can the 2-hydroxyisobutyryl-HIST1H1C (K25) antibody help investigate this function?

Research has revealed a critical role for HIST1H1C in regulating autophagy, particularly in the context of diabetic retinopathy. Studies have demonstrated that:

• Overexpression of HIST1H1C upregulates autophagy-related proteins (ATG12-ATG5 complex, ATG7, ATG3) and increases LC3B-I to LC3B-II conversion, which are key indicators of enhanced autophagy .

• HIST1H1C mechanistically promotes autophagy by upregulating SIRT1 and HDAC1, which maintain the deacetylation status of H4K16, leading to increased expression of autophagy-related genes .

• In diabetic retinopathy models, increased HIST1H1C levels correlate with elevated autophagy markers .

The 2-hydroxyisobutyryl-HIST1H1C (K25) antibody can help investigate whether this specific modification alters HIST1H1C's ability to regulate autophagy. Researchers can use this antibody in combination with autophagy markers to determine if 2-hydroxyisobutyrylation at K25 enhances or inhibits HIST1H1C's autophagy-promoting functions. By comparing the levels of 2-hydroxyisobutyryl-HIST1H1C (K25) with autophagy markers in normal versus disease states (such as diabetes), researchers can establish correlations between this specific modification and autophagy regulation .

How does HIST1H1C contribute to antiviral immunity and what experimental designs would be appropriate to study this using the K25 antibody?

Recent research has uncovered that HIST1H1C plays a significant role in antiviral immunity. Specifically:

• HIST1H1C has been found to inhibit Encephalomyocarditis virus (EMCV) replication by enhancing the production of type I interferons .

• Mechanistically, HIST1H1C upregulates the expression of proteins in the MDA5 signaling pathway and interacts directly with MDA5 and IRF3 during viral infection .

• HIST1H1C facilitates EMCV-induced phosphorylation and nuclear translocation of IRF3, a critical step in the interferon response .

To study the potential role of 2-hydroxyisobutyryl modification at K25 in this antiviral function, researchers could design experiments such as:

• Comparative immunoprecipitation studies: Use the 2-hydroxyisobutyryl-HIST1H1C (K25) antibody to immunoprecipitate modified HIST1H1C from virus-infected versus uninfected cells to determine if viral infection alters this specific modification.

• Co-localization experiments: Perform immunofluorescence using both the 2-hydroxyisobutyryl-HIST1H1C (K25) antibody and antibodies against antiviral signaling components (MDA5, IRF3) to determine if the modified form of HIST1H1C specifically interacts with these factors.

• Mutational analysis: Compare the antiviral activity of wild-type HIST1H1C versus a K25R mutant (which cannot be modified at this position) to determine if this specific site is critical for the protein's antiviral function .

How can the 2-hydroxyisobutyryl-HIST1H1C (K25) antibody be integrated into ChIP-seq workflows to study epigenome-wide effects?

While standard ChIP-seq protocols can be adapted for use with the 2-hydroxyisobutyryl-HIST1H1C (K25) antibody, several specialized considerations should be implemented:

• Optimization of crosslinking conditions: Since linker histones like HIST1H1C have different DNA binding properties than core histones, crosslinking conditions may need to be optimized. A dual crosslinking approach using both formaldehyde (1-2%) and a protein-protein crosslinker like DSG (disuccinimidyl glutarate) may improve capture of linker histone associations.

• Chromatin fragmentation: Linker histones occupy regions between nucleosomes, so optimal fragment sizes may differ from standard ChIP-seq protocols. Aim for fragments between 150-300 bp through careful sonication optimization.

• Antibody validation controls:

  • Include immunoprecipitation with a pan-HIST1H1C antibody as a reference dataset

  • Perform peptide competition assays using both modified and unmodified peptides to confirm specificity

  • Include technical controls using cells where HIST1H1C has been knocked down

• Data analysis considerations:

  • Expected binding patterns may differ from core histone modifications, with enrichment potentially occurring at linker regions and regulatory elements

  • Compare with datasets for other histone modifications to identify unique patterns associated with 2-hydroxyisobutyryl-K25

  • Integrate with transcriptomic data to correlate modification patterns with gene expression changes

What are the key considerations for validating the specificity of the 2-hydroxyisobutyryl-HIST1H1C (K25) antibody in experimental systems?

Ensuring antibody specificity is critical for accurate interpretation of results, particularly with histone modification antibodies. For the 2-hydroxyisobutyryl-HIST1H1C (K25) antibody, consider these validation approaches:

• Peptide competition assays:

  • Perform immunostaining or western blots in the presence of:

    • The specific 2-hydroxyisobutyryl-K25 modified peptide (should block signal)

    • Unmodified K25 peptide (should not block signal)

    • Peptides with other modifications at K25 (should not block signal)

    • 2-hydroxyisobutyryl modified peptides from other lysine residues of HIST1H1C (should not block signal)

• Genetic validation:

  • Test antibody reactivity in systems where:

    • HIST1H1C is knocked down or knocked out (should see reduced or absent signal)

    • K25 is mutated to arginine (should see absent signal specifically for the K25 modification)

    • Enzymes responsible for 2-hydroxyisobutyrylation are manipulated (should see altered signal intensity)

• Cross-reactivity assessment:

  • Test against recombinant HIST1H1C with defined modifications

  • Evaluate potential cross-reactivity with similar modifications (e.g., acetylation, butyrylation) at the K25 position

  • Assess reactivity against other H1 variants that have similar sequences around their K25-equivalent residues

How can the 2-hydroxyisobutyryl-HIST1H1C (K25) antibody be used to investigate diabetic retinopathy mechanisms?

Diabetic retinopathy research has identified HIST1H1C as a potential therapeutic target, with studies showing its role in promoting autophagy, inflammation, and cell toxicity in retinal cells. The 2-hydroxyisobutyryl-HIST1H1C (K25) antibody can be instrumental in expanding this research:

• Modification changes in disease progression:

  • Compare levels of 2-hydroxyisobutyryl-K25 modification in retinal tissues from diabetic versus non-diabetic models

  • Track changes in this modification throughout disease progression using immunohistochemistry on retinal sections

  • Correlate modification levels with disease severity markers

• Functional studies in retinal cells:

  • Examine whether high glucose conditions alter 2-hydroxyisobutyryl-K25 levels in cultured retinal cells

  • Investigate if this specific modification affects HIST1H1C's ability to promote autophagy and inflammation in retinal cells

  • Use the antibody in ChIP experiments to identify genes whose regulation is affected by this modification in diabetic conditions

• Intervention studies:

  • Monitor changes in 2-hydroxyisobutyryl-K25 levels following experimental treatments for diabetic retinopathy

  • Investigate whether drugs that affect histone modifications can alter the levels of this specific modification and improve disease outcomes

  • Use the antibody to screen potential compounds that might specifically target this modification

What mechanisms might regulate 2-hydroxyisobutyrylation of HIST1H1C at K25, and how can these be experimentally investigated?

The regulation of 2-hydroxyisobutyrylation at HIST1H1C K25 likely involves specific enzymes and metabolic conditions, though the exact mechanisms remain to be fully elucidated. Several experimental approaches can help investigate these regulatory mechanisms:

• Metabolic regulation:

  • Examine how cellular metabolic states (glucose levels, mitochondrial function) affect 2-hydroxyisobutyryl-K25 levels

  • Measure levels of 2-hydroxyisobutyryl-CoA under different conditions and correlate with K25 modification levels

  • Investigate whether conditions that alter the NAD+/NADH ratio affect this modification

• Enzyme identification:

  • Perform screening experiments with candidate transferases and deacetylases to identify enzymes that can add or remove this modification

  • Use mass spectrometry following immunoprecipitation with the 2-hydroxyisobutyryl-HIST1H1C (K25) antibody to identify proteins that interact with the modified form

  • Conduct gain- and loss-of-function studies with candidate enzymes and measure effects on K25 modification levels

• Signaling pathways:

  • Investigate how stress conditions (oxidative stress, ER stress) affect 2-hydroxyisobutyryl-K25 levels

  • Examine the roles of known epigenetic regulators (sirtuins, HDACs) in controlling this modification

  • Study how cell cycle progression and differentiation states impact the levels of this modification

What are the common technical challenges when using the 2-hydroxyisobutyryl-HIST1H1C (K25) antibody and how can they be addressed?

When working with the 2-hydroxyisobutyryl-HIST1H1C (K25) antibody, researchers may encounter several technical challenges that can be addressed with specific strategies:

• High background in immunostaining:

  • Increase blocking time using 3-5% BSA or 5-10% normal serum

  • Optimize antibody dilution (try 1:100 for initial tests, then adjust)

  • Include additional washing steps with 0.1% Tween-20 in PBS

  • Pre-absorb antibody with unmodified histone extract

• Weak or no signal in western blots:

  • Ensure proper extraction of nuclear proteins using acid extraction methods

  • Include deacetylase inhibitors in all buffers to preserve modifications

  • Transfer proteins to PVDF rather than nitrocellulose membranes

  • Use higher antibody concentration (1:100 instead of 1:1000) and extend incubation time to overnight at 4°C

• Cross-reactivity concerns:

  • Include competitive blocking with specific and non-specific peptides

  • Perform parallel experiments with pan-HIST1H1C antibody to confirm protein presence

  • Include samples with HIST1H1C knockdown as negative controls

  • Validate signals with orthogonal techniques like mass spectrometry

• Storage and handling:

  • Aliquot antibody upon receipt to avoid repeated freeze-thaw cycles

  • Store at -80°C for long-term storage

  • Add carrier protein (0.1% BSA) if diluting for storage

  • Follow manufacturer guidelines for the specific antibody formulation

How can researchers quantitatively compare levels of 2-hydroxyisobutyryl-HIST1H1C (K25) across different experimental conditions?

Quantitative comparison of 2-hydroxyisobutyryl-HIST1H1C (K25) levels requires careful experimental design and appropriate normalization strategies:

• Western blot quantification:

  • Always include loading controls (total HIST1H1C or other stable nuclear proteins)

  • Prepare standard curves using recombinant modified proteins when available

  • Use digital imaging systems with linear dynamic range

  • Express results as the ratio of modified to total HIST1H1C

• Immunofluorescence quantification:

  • Apply consistent image acquisition parameters across all samples

  • Measure nuclear fluorescence intensity using software like ImageJ

  • Use DAPI or total HIST1H1C staining for normalization

  • Include internal controls on each slide for inter-slide normalization

• ChIP-qPCR approaches:

  • Normalize enrichment to input DNA

  • Include IgG controls to account for non-specific binding

  • Perform parallel ChIP with antibodies against total HIST1H1C

  • Express results as enrichment relative to a genomic region known to lack HIST1H1C or as a ratio of modified/total HIST1H1C

• Mass spectrometry validation:

  • Use stable isotope labeling when possible

  • Analyze ratio of modified to unmodified peptides containing K25

  • Include standard peptides for absolute quantification

  • Consider analyzing multiple histone modifications simultaneously for deeper insights