2-hydroxyisobutyryl-HIST1H1C (K168) Antibody

What is 2-hydroxyisobutyryl-HIST1H1C (K168) Antibody and what specific modification does it detect?

2-hydroxyisobutyryl-HIST1H1C (K168) Antibody is a polyclonal antibody raised in rabbits that specifically recognizes the 2-hydroxyisobutyryl post-translational modification at lysine 168 (K168) of human Histone H1.2 (HIST1H1C). This antibody was generated using a peptide sequence around the 2-hydroxyisobutyryl-Lys (168) site derived from Human Histone H1.2 . The antibody's specificity for this particular histone modification makes it a valuable tool for studying epigenetic mechanisms involved in chromatin regulation and gene expression.

What is the molecular background of the HIST1H1C protein targeted by this antibody?

HIST1H1C encodes Histone H1.2, which belongs to the linker histone H1 family. Also known as H1c, H1d, or H1s-1, this protein has a calculated molecular weight of 21 kDa but is typically observed at 32-33 kDa in experimental conditions . Histone H1.2 plays critical roles in higher-order chromatin structure and transcriptional regulation. The protein's UniProt ID is P16403, and its NCBI Gene ID is 3006 . The 2-hydroxyisobutyrylation at K168 represents a specific post-translational modification that may regulate the protein's function in chromatin dynamics and gene expression patterns.

What are the primary research applications for this antibody?

The 2-hydroxyisobutyryl-HIST1H1C (K168) Antibody has been validated for multiple research applications:

The antibody can be used to study epigenetic and nuclear signaling pathways, particularly in the context of histone modifications and their role in gene regulation .

What is the optimal sample preparation protocol for immunofluorescence studies using this antibody?

For optimal immunofluorescence results with 2-hydroxyisobutyryl-HIST1H1C (K168) Antibody, implement the following validated protocol:

• Fix cells in 4% formaldehyde solution for proper preservation of cellular structure and epitopes

• Permeabilize with 0.2% Triton X-100 to allow antibody access to nuclear proteins

• Block with 10% normal goat serum for 30 minutes at room temperature to reduce non-specific binding

• Incubate with primary antibody (diluted 1:10-1:100 in 1% BSA) overnight at 4°C

• Wash thoroughly with PBS (3-5 times)

• Incubate with a compatible fluorescent secondary antibody (e.g., Alexa Fluor 488-conjugated AffiniPure Goat Anti-Rabbit IgG)

• Counterstain nuclei with DAPI for orientation

• Mount and visualize using fluorescence microscopy

This protocol has been specifically optimized for detection of the 2-hydroxyisobutyryl modification on HIST1H1C in cultured cells, as demonstrated in validation experiments using HeLa cells treated with 30mM sodium butyrate .

How should researchers modify cell treatment to enhance detection of 2-hydroxyisobutyryl-HIST1H1C?

To enhance detection of 2-hydroxyisobutyrylated HIST1H1C, pretreatment of cells with histone deacetylase (HDAC) inhibitors can significantly increase signal intensity. Based on validation data, researchers should:

• Treat cells with 30mM sodium butyrate for 4 hours prior to fixation

• Maintain consistent treatment times between experiments to ensure reproducibility

• Consider cell-type specific optimization as different cell lines may require adjusted treatment concentrations

• Include untreated controls to establish baseline modification levels

This pretreatment increases global histone acylation levels, including 2-hydroxyisobutyrylation, making detection more robust. The optimized protocol has been validated for HeLa cells but may require adjustment for other cell types.

What are the recommended storage and handling conditions to maintain antibody performance?

To preserve antibody functionality and ensure consistent experimental results:

• Upon receipt, aliquot the antibody to avoid repeated freeze-thaw cycles

• Store at -20°C or -80°C according to manufacturer recommendations

• Avoid more than 3 freeze-thaw cycles which can degrade antibody performance

• When removing from storage, thaw on ice and centrifuge briefly before opening

• Return unused portion to -20°C immediately after use

• Note the storage buffer composition: 50% Glycerol, 0.01M PBS, pH 7.4 with 0.03% Proclin 300 as preservative

Following these guidelines will maintain antibody integrity for at least one year from the date of receipt when stored properly.

How does 2-hydroxyisobutyryl-HIST1H1C (K168) modification differ from other histone post-translational modifications in functional impact?

The 2-hydroxyisobutyrylation (2-hib) of HIST1H1C at K168 represents a distinct epigenetic mark from more extensively studied modifications like acetylation and methylation. Current research indicates:

• 2-hydroxyisobutyrylation creates a bulkier modification than acetylation, potentially causing more significant structural changes to chromatin

• Unlike methylation which can occur at multiple levels (mono-, di-, tri-), 2-hib is a binary modification

• 2-hib modifications appear to be associated with active gene transcription, particularly during cell proliferation

• The enzymatic machinery regulating 2-hib addition and removal is less well characterized than that for acetylation

• 2-hib modifications on H1 variants like HIST1H1C may specifically regulate linker histone positioning and mobility

This specific modification at K168 of HIST1H1C provides a unique research focus for understanding specialized epigenetic regulation mechanisms that control chromatin architecture and accessibility .

What experimental approaches can differentiate effects of HIST1H1C 2-hydroxyisobutyrylation from other histone H1 variants?

To specifically study the effects of 2-hydroxyisobutyrylation on HIST1H1C (K168) versus other histone variants, researchers should implement:

• Combinatorial immunoprecipitation approach: Use the 2-hydroxyisobutyryl-HIST1H1C (K168) Antibody for primary IP followed by immunoblotting with antibodies specific to different H1 variants to determine modification distribution

• Genomic localization via ChIP-seq:

  • Perform ChIP-seq with 2-hydroxyisobutyryl-HIST1H1C (K168) Antibody

  • Compare enrichment patterns with ChIP-seq data from other H1 variant-specific antibodies

  • Analyze genomic regions where HIST1H1C 2-hydroxyisobutyrylation is uniquely enriched

• CRISPR/Cas9 modification approach:

  • Generate K168R mutation specifically in the HIST1H1C gene to prevent 2-hydroxyisobutyrylation

  • Compare phenotypes and gene expression profiles with wild-type cells

  • Use the CRISPR/Cas9 system similar to that employed in HIST1H1C knockout studies

• Mass spectrometry verification:

  • Immunoprecipitate with 2-hydroxyisobutyryl-HIST1H1C (K168) Antibody

  • Perform LC-MS/MS analysis to confirm specificity and identify potentially co-occurring modifications

These approaches can dissect variant-specific functions of 2-hydroxyisobutyrylation in chromatin regulation.

What role might 2-hydroxyisobutyrylated HIST1H1C play in hepatocarcinogenesis based on recent studies?

Recent research indicates that HIST1H1C (Histone H1.2) has significant implications for hepatocarcinogenesis, though the specific contribution of its 2-hydroxyisobutyrylation remains under investigation. Studies using HIST1H1C knockout models (Hist1h1c KO mice) generated via CRISPR/Cas9 have provided insights into its function in liver cancer development:

• HIST1H1C appears to regulate signal transduction pathways involved in hepatocellular carcinoma (HCC) progression

• In DEN-induced HCC mouse models, HIST1H1C expression patterns show significant correlation with cancer development

• Immunohistochemical studies have revealed interactions between HIST1H1C and other cancer-related markers including Ki-67, F4/80, CD3, and Ly6G

• Analysis of human HCC tissue microarrays comprising 15 pairs of tumor and paratumor tissues showed correlations between HIST1H1C expression and phosphorylated STAT3 (Y705)

The 2-hydroxyisobutyrylation at K168 may represent a regulatory mechanism affecting HIST1H1C's role in these pathways, potentially influencing its interaction with transcription factors or chromatin remodeling complexes that drive oncogenic programs. This presents an important area for future research using the 2-hydroxyisobutyryl-HIST1H1C (K168) Antibody.

What are the most common technical challenges when using 2-hydroxyisobutyryl-HIST1H1C (K168) Antibody in immunocytochemistry?

When performing immunocytochemistry with 2-hydroxyisobutyryl-HIST1H1C (K168) Antibody, researchers commonly encounter these technical challenges and their solutions:

Based on validation data, the antibody performs optimally when used in the BondTM system for ICC applications and visualized using an HRP conjugated SP system after appropriate blocking and permeabilization .

How can researchers validate the specificity of signals detected using this antibody?

To rigorously validate the specificity of 2-hydroxyisobutyryl-HIST1H1C (K168) Antibody signals, implement these experimental controls:

• Peptide competition assay:

  • Pre-incubate antibody with excess immunizing peptide (2-hydroxyisobutyryl-K168 containing peptide)

  • Process samples in parallel with competed and non-competed antibody

  • Specific signals should be eliminated in the competed samples

• Knockout/knockdown validation:

  • Use CRISPR/Cas9-mediated HIST1H1C knockout models similar to those described in hepatocarcinogenesis studies

  • Compare staining patterns between wild-type and knockout samples

  • Specific signal should be absent in knockout samples

• Enzymatic removal of modification:

  • Treat fixed samples with deacylases prior to immunostaining

  • Compare treated and untreated samples

  • Specific signal should be reduced in enzyme-treated samples

• Dual detection methods:

  • Combine IF with mass spectrometry validation

  • Immunoprecipitate with the antibody and verify the presence of the modification by MS/MS

• Cross-validation with orthogonal antibodies:

  • Compare staining patterns with antibodies targeting the same modification but from different manufacturers

  • Consistent patterns support specificity

These approaches provide multiple lines of evidence to confirm signal specificity.

How does 2-hydroxyisobutyrylation of HIST1H1C integrate with the broader histone code hypothesis?

The identification of 2-hydroxyisobutyrylation on HIST1H1C at K168 expands the complexity of the histone code hypothesis in several important ways:

• As a relatively newly characterized modification, 2-hydroxyisobutyrylation adds another layer to the combinatorial pattern of histone modifications that regulate gene expression

• Unlike the extensively studied core histones (H2A, H2B, H3, H4), modifications on linker histones like HIST1H1C have received less attention, despite their critical role in higher-order chromatin organization

• The specific location of K168 in HIST1H1C may regulate:

  • Interactions with DNA at linker regions

  • Associations with other nuclear proteins

  • Accessibility of chromatin to transcription factors

• 2-hydroxyisobutyrylation represents a metabolic-epigenetic connection, potentially linking cellular metabolism (through 2-hydroxyisobutyryl-CoA levels) to chromatin regulation

• The combination of this modification with other PTMs on HIST1H1C may create specific "chromatin states" that direct specialized transcriptional outcomes

Future research using the 2-hydroxyisobutyryl-HIST1H1C (K168) Antibody will help define the "readers," "writers," and "erasers" of this modification and its position within the broader histone code framework.

What methodological approaches can integrate 2-hydroxyisobutyryl-HIST1H1C data with other omics datasets?

To maximize the research value of 2-hydroxyisobutyryl-HIST1H1C (K168) data, researchers should consider these integrative methodological approaches:

• Multi-omics integration workflow:

  • Perform ChIP-seq using 2-hydroxyisobutyryl-HIST1H1C (K168) Antibody

  • Generate parallel RNA-seq and ATAC-seq data from matching samples

  • Integrate datasets using computational tools like HOMER, ChIPseeker, and multiomics visualization platforms

  • Identify genomic regions where 2-hydroxyisobutyrylation correlates with transcriptional activity and chromatin accessibility

• Temporal dynamics analysis:

  • Conduct time-course experiments following stimuli that induce epigenetic reprogramming

  • Track changes in 2-hydroxyisobutyryl-HIST1H1C levels in relation to transcriptional responses

  • Apply mathematical modeling to identify cause-effect relationships

• Single-cell epigenomics approach:

  • Adapt 2-hydroxyisobutyryl-HIST1H1C (K168) Antibody for CUT&Tag or related single-cell compatible methodologies

  • Integrate with single-cell RNA-seq to correlate modification presence with gene expression at individual cell level

  • Identify cell populations with distinctive 2-hydroxyisobutyrylation patterns

• Proteomic interaction network:

  • Use the antibody for immunoprecipitation followed by mass spectrometry

  • Identify proteins that interact with 2-hydroxyisobutyrylated HIST1H1C

  • Construct protein-protein interaction networks to map functional pathways

These integrative approaches will place 2-hydroxyisobutyrylation of HIST1H1C in broader regulatory contexts and reveal its functional significance.

What are the potential therapeutic implications of targeting 2-hydroxyisobutyrylated HIST1H1C in disease models?

Emerging research on HIST1H1C's role in diseases like hepatocellular carcinoma suggests several therapeutic implications for targeting its 2-hydroxyisobutyrylation:

• Cancer therapy development:

  • Given HIST1H1C's role in hepatocarcinogenesis , targeting its 2-hydroxyisobutyrylation could disrupt cancer-promoting chromatin states

  • The 2-hydroxyisobutyryl-HIST1H1C (K168) Antibody could help identify patient populations with aberrant modification levels for precision medicine approaches

  • Development of small molecules that inhibit the enzymatic machinery placing this modification could represent a novel therapeutic strategy

• Epigenetic drug screening platform:

  • Use the antibody to develop high-throughput screening assays for compounds that modulate 2-hydroxyisobutyrylation levels

  • Screen existing epigenetic drugs for off-target effects on this modification

  • Identify combinatorial approaches that target multiple histone modifications simultaneously

• Diagnostic biomarker potential:

  • Evaluate 2-hydroxyisobutyryl-HIST1H1C levels in patient samples across various diseases

  • Correlate modification patterns with clinical outcomes and treatment responses

  • Develop immunohistochemistry-based diagnostic panels incorporating this antibody for precision pathology

• Metabolic-epigenetic interventions:

  • Investigate dietary or pharmacological approaches that modulate cellular metabolism to affect 2-hydroxyisobutyryl-CoA levels

  • Target the metabolic pathways that produce the 2-hydroxyisobutyryl donor molecules

These therapeutic approaches would benefit from further mechanistic studies using the 2-hydroxyisobutyryl-HIST1H1C (K168) Antibody to understand the precise role of this modification in health and disease.