2-hydroxyisobutyryl-HIST1H1C (K210) Antibody

What is HIST1H1C and what is its function in chromatin structure?

HIST1H1C (Histone H1.2) is a linker histone that interacts with DNA between nucleosomes, facilitating chromatin condensation into higher-order fibers. This histone affects nucleosome spacing, chromatin remodeling, and DNA methylation, thereby modulating gene expression. It plays a vital role in the formation, regulation, and maintenance of correct chromatin high structure . As a member of the H1 histone family, HIST1H1C contributes to chromatin compaction and is essential for proper nuclear architecture and genomic stability.

How does HIST1H1C differ from other H1 histone variants?

HIST1H1C (H1.2) is one of several somatic H1 variants (H1.1-H1.5) that show tissue-specific and developmental expression patterns. Research from hepatocellular carcinoma studies has shown that H1C expression, but not other somatic H1 variants, was significantly altered in HCC patients . The calculated molecular weight of HIST1H1C is 21 kDa, while the observed molecular weight in laboratory conditions is 32-33 kDa . Different H1 variants may have specialized functions in chromatin organization and gene regulation, with HIST1H1C playing roles in processes such as senescence-associated heterochromatin formation .

What are the optimal applications and dilutions for the 2-hydroxyisobutyryl-HIST1H1C (K109) antibody?

The 2-hydroxyisobutyryl-HIST1H1C (K109) antibody can be used in multiple applications with the following recommended dilutions:

For Proteintech's HIST1H1C antibody (19649-1-AP), the following dilutions are recommended:

It is important to note that these antibodies should be titrated in each testing system to obtain optimal results, as outcomes may be sample-dependent .

What cell types and tissues have been validated for 2-hydroxyisobutyryl-HIST1H1C (K109) antibody use?

The 2-hydroxyisobutyryl-HIST1H1C antibody has been validated to detect endogenous levels of HIST1H1C of human origin . For the related HIST1H1C antibody (19649-1-AP), positive Western blot detection has been confirmed in:

• L02 cells

• Human testis tissue

• Jurkat cells

• MCF-7 cells

• A375 cells

• Mouse thymus tissue

Positive immunoprecipitation has been detected in HeLa cells. Positive IHC detection has been observed in human ovary tumor tissue and human normal colon. Positive IF/ICC detection has been confirmed in HeLa cells and HepG2 cells .

What antigen retrieval methods are recommended for immunohistochemistry with HIST1H1C antibodies?

For optimal immunohistochemical detection of HIST1H1C, the following antigen retrieval methods are recommended:

• Primary method: TE buffer pH 9.0

• Alternative method: Citrate buffer pH 6.0

For tissue microarray analysis of human HCC samples, researchers have used overnight incubation at room temperature with antibodies for H1.2 or p-STAT3 Y705. Positive staining can be visualized using 3,3'-diaminobenzidine substrate following the ABC kit protocol .

How can researchers design ChIP experiments to investigate 2-hydroxyisobutyryl-HIST1H1C binding to specific genomic regions?

When designing ChIP experiments for 2-hydroxyisobutyryl-HIST1H1C, researchers should:

• Select appropriate target regions: Previous studies have focused on three different regions of human H1C or mouse H1c promoter ranging from -2000 bp to the transcription start site (TSS) .

• Use proper controls: Include input DNA, IgG controls, and potentially antibodies against unmodified HIST1H1C.

• Validate antibody specificity: Ensure the antibody specifically recognizes 2-hydroxyisobutyryl modification at K109, with minimal cross-reactivity.

• Consider fixation conditions: Optimize formaldehyde concentration and fixation time to preserve protein-DNA interactions while allowing antibody access.

• Sonication parameters: Adjust sonication conditions to obtain DNA fragments of 200-500 bp for optimal resolution.

• Analyze potential binding motifs: Bioinformatic tools like JASPAR can be used to analyze transcription factor binding sites within promoters of interest, as was done for STAT3 binding sites within human H1C or mouse H1c promoters .

What are the challenges in interpreting 2-hydroxyisobutyryl-HIST1H1C data in the context of hepatocellular carcinoma research?

Interpreting 2-hydroxyisobutyryl-HIST1H1C data in hepatocellular carcinoma (HCC) research presents several challenges:

How should researchers approach the study of 2-hydroxyisobutyryl-HIST1H1C in senescence-associated heterochromatin formation?

When studying 2-hydroxyisobutyryl-HIST1H1C in senescence-associated heterochromatin foci (SAHF) formation, researchers should consider:

• Temporal dynamics: Monitor the dynamics of 2-hydroxyisobutyryl-HIST1H1C during the induction of cellular senescence, using time-course experiments to determine when changes occur.

• Co-localization studies: Perform immunofluorescence to examine co-localization of 2-hydroxyisobutyryl-HIST1H1C with known SAHF markers such as H3K9me3, HP1, and HMGA proteins.

• Interaction partners: Use co-immunoprecipitation to identify protein-protein interactions between 2-hydroxyisobutyryl-HIST1H1C and other chromatin regulators involved in SAHF formation, such as CABIN1 and UBN1 .

• Functional studies: Employ RNAi or CRISPR-Cas9 approaches to modulate HIST1H1C levels or specifically target the enzyme responsible for K109 2-hydroxyisobutyrylation to assess functional consequences on SAHF formation.

• Genomic distribution: Conduct ChIP-seq to map the genome-wide distribution of 2-hydroxyisobutyryl-HIST1H1C during senescence, comparing with the distribution of other histone modifications and chromatin factors.

What are the common issues in Western blot detection of 2-hydroxyisobutyryl-HIST1H1C and how can they be resolved?

When performing Western blot for 2-hydroxyisobutyryl-HIST1H1C detection, researchers may encounter several challenges:

• Molecular weight discrepancy: The calculated molecular weight of HIST1H1C is 21 kDa, while the observed molecular weight is 32-33 kDa . This difference can cause confusion in band identification. Solution: Always include positive controls and validate with additional techniques.

• Non-specific binding: Multiple bands may appear due to cross-reactivity with other histone variants or modifications. Solution: Optimize antibody dilution (recommended range 1:100-1:1000 for 2-hydroxyisobutyryl-HIST1H1C K109 antibody) and increase blocking time/concentration.

• Weak signal: 2-hydroxyisobutyrylation may be present at low abundance. Solution: Increase protein loading (30-50 μg), enhance antibody concentration, or use signal enhancement systems.

• Background issues: High background can obscure specific signals. Solution: Increase washing duration/frequency and optimize blocking conditions.

• Sample preparation: Histone modifications can be lost during extraction. Solution: Use specialized histone extraction protocols with deacetylase and phosphatase inhibitors to preserve modifications.

How can immunofluorescence protocols be optimized for simultaneous detection of 2-hydroxyisobutyryl-HIST1H1C and other nuclear markers?

To optimize immunofluorescence protocols for simultaneous detection of 2-hydroxyisobutyryl-HIST1H1C and other nuclear markers:

• Fixation optimization:

  • Use 4% paraformaldehyde for 10-15 minutes at room temperature

  • For better nuclear antigen accessibility, consider adding a brief methanol treatment (-20°C for 5 minutes)

• Permeabilization:

  • Use 0.2-0.5% Triton X-100 for 10 minutes for optimal nuclear access

  • For co-staining with membrane markers, reduce Triton X-100 concentration to 0.1%

• Antibody dilutions:

  • For 2-hydroxyisobutyryl-HIST1H1C (K109) antibody: Use at 1:20-1:200 dilution

  • For HIST1H1C antibody (19649-1-AP): Use at 1:50-1:500 dilution

• Sequential staining:

  • For antibodies from the same species, use sequential staining with a blocking step between antibodies

  • Consider using directly conjugated antibodies for one marker to avoid cross-reactivity

• Controls to include:

  • Single-stained controls to assess bleed-through

  • Secondary antibody-only controls to check for non-specific binding

  • Positive controls in cells known to express high levels of HIST1H1C (HeLa or HepG2 cells)

What considerations should be made when comparing different histone H1 variant antibodies in research studies?

When comparing different histone H1 variant antibodies in research studies, several important considerations should be addressed:

• Specificity verification:

  • Conduct validation using knockout/knockdown cells

  • Test antibodies on purified recombinant H1 variants

  • Perform peptide competition assays to confirm epitope specificity

• Cross-reactivity assessment:

  • Test antibodies against all H1 variants (H1.1-H1.5) using purified proteins

  • Sequence alignment analysis to identify regions of high similarity between variants

  • Document any observed cross-reactivity in experimental records

• Modification-specific recognition:

  • Determine whether antibodies recognize specific post-translational modifications

  • For 2-hydroxyisobutyryl-HIST1H1C antibodies, confirm specificity for the K109 site

  • Check for cross-reactivity with other lysine modifications (acetylation, methylation, etc.)

• Application-specific performance:

  • Compare antibody performance across different applications (WB, IHC, IF, ChIP)

  • Document optimal dilutions for each application across different antibodies

  • Different antibodies may perform better in different applications:

    • For Western blot: 1:500-1:3000 (HIST1H1C antibody) vs. 1:100-1:1000 (2-hydroxyisobutyryl-HIST1H1C)

    • For IHC: 1:100-1:600 (HIST1H1C antibody)

    • For IF: 1:50-1:500 (HIST1H1C antibody) vs. 1:20-1:200 (2-hydroxyisobutyryl-HIST1H1C)

• Reproducibility between lots:

  • Test multiple antibody lots to assess consistency

  • Document lot numbers and observed variations in experimental notes

  • Consider using monoclonal antibodies for higher consistency when available

How can 2-hydroxyisobutyryl-HIST1H1C antibodies be used to study epigenetic changes in hepatocellular carcinoma?

2-hydroxyisobutyryl-HIST1H1C antibodies offer valuable tools for studying epigenetic changes in hepatocellular carcinoma (HCC) through multiple approaches:

• Tissue microarray analysis:

  • Human HCC tissue microarrays can be analyzed using IHC with H1.2 antibodies

  • Semiquantitative scoring systems (0-5) corresponding to percentage of positively stained cells (0% to 80-100%) can quantify expression levels

  • Comparative analysis between tumor and paratumor tissues reveals significant differences in H1.2 expression

• Correlation with clinical parameters:

  • H1.2 staining can be correlated with patient survival, tumor stage, and other clinical parameters

  • Studies have shown significantly higher nuclear H1.2 staining in human HCC samples compared to paratumor tissues

• Signaling pathway analysis:

  • Co-staining with pathway markers like p-STAT3 Y705

  • Pearson's correlation coefficient analysis between H1.2 and p-STAT3 Y705 levels reveals regulatory relationships

  • Research has demonstrated that H1.2 promotes hepatocarcinogenesis by regulating signal transducer and activator of transcription pathways

• Animal models:

  • In DEN-treated mice (a model for HCC), significantly increased H1.2 levels were found in tumors compared to liver tissue from age-matched controls

  • Higher H1.2 levels were observed in tumors compared with paratumor tissues

• Subcellular localization:

  • Co-staining with nuclear (DAPI) or mitochondrial (TIM23) markers to determine compartmentalization of H1.2

  • Most H1.2 staining is found in nuclei rather than mitochondria in HCC samples

What experimental designs can elucidate the role of 2-hydroxyisobutyryl-HIST1H1C in chromatin remodeling during cellular differentiation?

To investigate the role of 2-hydroxyisobutyryl-HIST1H1C in chromatin remodeling during cellular differentiation, researchers can implement these experimental designs:

• Time-course analysis during differentiation:

  • Monitor 2-hydroxyisobutyryl-HIST1H1C levels at multiple timepoints during differentiation using Western blot and immunofluorescence

  • Compare with markers of differentiation and other histone modifications

  • Correlate changes with chromatin accessibility using techniques like ATAC-seq

• ChIP-seq analysis:

  • Map genomic distribution of 2-hydroxyisobutyryl-HIST1H1C before, during, and after differentiation

  • Identify differentially bound regions and correlate with gene expression changes

  • Compare with binding patterns of chromatin remodelers and other histone modifications

  • Analyze potential binding sites within gene promoters using bioinformatic tools like JASPAR

• Functional perturbation studies:

  • Use CRISPR-Cas9 to generate K109 mutants that cannot be 2-hydroxyisobutyrylated

  • Assess effects on differentiation potential and chromatin structure

  • Employ inducible systems to temporally control HIST1H1C expression or modification

• Interaction mapping:

  • Conduct co-immunoprecipitation assays to identify proteins interacting with 2-hydroxyisobutyryl-HIST1H1C during differentiation

  • Perform proximity ligation assays to visualize interactions in situ

  • Compare interaction networks at different differentiation stages

• Super-resolution microscopy:

  • Visualize chromatin compaction states using techniques like STORM or PALM

  • Co-localize 2-hydroxyisobutyryl-HIST1H1C with heterochromatin or euchromatin markers

  • Quantify spatial changes in chromatin organization throughout differentiation