HIST1H1C (Ab-105) Antibody

What is HIST1H1C and what is its biological role?

HIST1H1C (also known as Histone H1.2, H1c, H1d, or H1F2) is a member of the H1 histone family that functions as a linker histone. It binds to DNA between nucleosomes, protecting an additional 20 base pairs of DNA and playing a fundamental role in promoting chromatin fiber condensation . Histone H1.2 acts as a regulator of individual gene transcription through chromatin remodeling, nucleosome spacing, and DNA methylation . Studies indicate that H1 histones limit nucleosome mobility, reduce transient exposure of DNA on the surface of nucleosomes, and can occlude transcription factor binding, suggesting a general repressive function in transcription regulation .

How does HIST1H1C differ from other H1 histone variants?

Despite high sequence homology between H1 variants (74-87% sequence similarity), each variant exhibits distinct functional properties . HIST1H1C (H1.2) has shown specific genome distribution patterns that differ from other variants. For instance, in studies using ChIP-seq, H1.2 (corresponding to mouse H1c) shows enrichment in major satellite repeats that constitute pericentromeres forming chromocentres with more stable and condensed chromatin conformation . The functional differences between variants are also evident in overexpression experiments, where different H1 variants produce distinct cellular phenotypes . The divergence in sequences between variants is primarily located at the amino and carboxy termini, which serve as the basis for generating variant-specific antibodies .

What are the specific characteristics of the HIST1H1C (Ab-105) antibody?

The HIST1H1C (Ab-105) antibody is a polyclonal antibody raised in rabbits that specifically targets a peptide sequence around site of Lysine 105 derived from Histone H1.2 . It's designed for use in ELISA and immunofluorescence applications with a recommended dilution of 1:1-1:10 for immunofluorescence . The antibody is purified using antigen affinity methods and is typically provided in a liquid form with a buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative .

What are the validated research applications for HIST1H1C (Ab-105) antibody?

Based on available data, the HIST1H1C (Ab-105) antibody has been validated for ELISA and immunofluorescence (IF) applications . Unlike some pan-H1 antibodies that show variable affinity between variants, variant-specific antibodies like this one show high selectivity without cross-reactivity with other H1 variants . When working with this antibody, researchers should consider using it for targeted applications such as:

• Detection of HIST1H1C in fixed cells and tissues via immunofluorescence

• Quantification of HIST1H1C levels via ELISA

• Potential applications in western blotting (though specific validation for this may be required)

How can I optimize ChIP experiments using the HIST1H1C antibody?

While the product information specifically mentions ELISA and IF applications, researchers interested in ChIP experiments with HIST1H1C should consider the following optimization protocol based on established H1 variant ChIP methodologies:

• Crosslinking: Perform formaldehyde crosslinking (1% for 10 minutes at room temperature) to preserve protein-DNA interactions.

• Chromatin preparation: Sonicate chromatin to generate fragments of 200-500 bp.

• Immunoprecipitation:

  • Use 2-5 μg of HIST1H1C antibody per ChIP reaction

  • Include appropriate negative controls (IgG, no antibody)

  • Incubate overnight at 4°C with rotation

• Washing and elution: Follow standard ChIP protocols with multiple stringent washes.

• qPCR validation: Perform real-time PCR using specific primers for known target regions.

Researchers have successfully performed ChIP-qPCR for H1 variants by correcting ChIP values by the corresponding input chromatin sample . For genome-wide analyses, ChIP can be combined with tiling arrays or high-resolution sequencing .

What controls should I include when validating antibody specificity?

Given the high sequence homology between H1 variants, validating antibody specificity is crucial. Consider the following controls:

• Western blot against purified recombinant H1 variants: Test against a panel of purified H1 variants (H1.0, H1.1, H1.2, H1.3, H1.4, H1.5, and H1X) to confirm specificity for HIST1H1C/H1.2.

• Peptide competition assay: Pre-incubate the antibody with excess immunizing peptide to confirm specific binding is blocked.

• Knockdown validation: Use siRNA or CRISPR to reduce HIST1H1C expression and confirm corresponding signal reduction.

• Comparison with tagged H1.2: Compare staining patterns with cells expressing tagged versions of H1.2 (e.g., HA-tagged recombinant H1.2) .

Research has shown that antibodies generated toward the tail domains of H1 could have varying specificity based on post-translational modification combinations present on the H1 molecule , so these validation steps are essential.

What are the major challenges in studying histone H1 variants?

Research on H1 histones faces several significant challenges:

• Limited availability of specific antibodies: The high sequence homology between variants (74-87%) makes it difficult to produce high-specificity antibodies for individual variants . The divergence in sequences is primarily located at the amino and carboxy termini, requiring partial identification of epitopes from these domains for variant distinction .

• Post-translational modifications interference: The amino and carboxy terminal tails of H1 variants are among the most abundantly modified sequences in the cell, with multiple simultaneous PTMs regularly identified . These modifications can alter antibody affinity and complicate interpretation of results.

• MS analysis limitations: Mass spectrometry analysis of H1 histones is challenging due to their unusual amino acid composition. Tryptic digestion generates very short peptides (5 amino acids or less) with low relative hydrophobicities, resulting in poor sequence coverage compared to standard proteins .

• Functional redundancy: Multiple H1 variants can compensate for each other's functions, making it difficult to determine variant-specific roles .

How can I distinguish between different H1 variants in my experiments?

To distinguish between highly similar H1 variants, consider these approaches:

• Use of variant-specific antibodies: Select antibodies targeting the divergent N-terminal regions, such as the HIST1H1C (Ab-105) antibody .

• Tagged recombinant H1 variants: Express HA-tagged or other epitope-tagged versions of H1 variants to enable specific detection .

• MS-based approaches: Despite challenges, optimized mass spectrometry protocols can distinguish between variants based on unique peptides.

• DamID technology: This has been successfully used for genomic mapping of human H1 variants (H1.1 to H1.5) in cellular models .

• Knockout/knockdown followed by complementation: Deplete endogenous H1 variants and complement with tagged versions to study variant-specific functions.

How do post-translational modifications affect HIST1H1C antibody recognition?

Post-translational modifications (PTMs) can significantly impact antibody recognition of HIST1H1C. The terminal tails of H1 variants are heavily modified, with multiple PTMs occurring simultaneously . For example, an antibody generated toward unmodified HIST1H1C might show reduced affinity when the target epitope contains phosphorylation, methylation, or other modifications.

If the antibody's epitope (around Lys-105) is subject to modification, consider these approaches:

• Verification with recombinant unmodified protein: Compare signal between recombinant (unmodified) and cellular (potentially modified) HIST1H1C.

• Pre-treatment with phosphatases or other enzymes: Remove specific modifications before antibody application if modifications are suspected to interfere.

• Cross-validation with modification-specific antibodies: Use antibodies specific for known modifications near the epitope to determine modification status.

• Cell cycle synchronization: Some H1 modifications are cell cycle-dependent, so synchronizing cells can provide more consistent results.

How can I use HIST1H1C antibodies to study chromatin compaction and gene regulation?

To investigate HIST1H1C's role in chromatin structure and gene regulation:

• ChIP-seq for genome-wide distribution: Perform ChIP-seq to identify genomic regions enriched for HIST1H1C binding. Studies have shown variant-specific distribution patterns that correlate with different chromatin states .

• Co-immunoprecipitation with chromatin remodelers: Identify protein interaction partners that may mediate HIST1H1C's effects on chromatin structure.

• Micrococcal nuclease (MNase) sensitivity assays: Compare nuclease sensitivity in wild-type cells versus cells with depleted or overexpressed HIST1H1C to assess effects on chromatin accessibility.

• Integration with transcriptomics: Correlate HIST1H1C binding patterns with gene expression data to identify regulatory relationships.

• Fluorescence recovery after photobleaching (FRAP): Study the dynamics of HIST1H1C binding to chromatin in living cells using fluorescently tagged H1.2.

Research has shown that H1 variants have distinct binding patterns, with H1.2 (mouse H1c) showing enrichment in major satellite repeats that constitute pericentromeres . Triple knockout of H1 variants leads to vastly increased transcription from major satellite repeats in parallel with shorter nucleosomal repeat length .

What approaches can be used to map the genome-wide distribution of HIST1H1C?

Several approaches have been successfully used to map H1 variant genomic distribution:

• ChIP-seq with specific antibodies: Using highly specific antibodies like HIST1H1C (Ab-105) for chromatin immunoprecipitation followed by sequencing .

• Tagged variant expression: Generate cell lines expressing tagged versions of HIST1H1C (e.g., HA-tag, FLAG-tag) for ChIP-seq analysis .

• DamID technology: This alternative to ChIP has been successfully used for genomic mapping of human H1 variants in IMR90 cells .

• CUT&RUN or CUT&Tag: These newer techniques may offer advantages over traditional ChIP for mapping histone variant distributions.

• Nucleosome positioning correlation: Combine H1.2 mapping with H3 distribution to understand the relationship between linker histone binding and nucleosome positioning .

Studies have revealed that H1 variants can be enriched in specific genomic regions. For instance, analysis of H1 variants in breast cancer cells showed that while all H1 variants occur across the genome, H1.2 has specific features both at promoters and genome-wide .

How does binding affinity of HIST1H1C compare to other H1 variants?

Quantitative analysis of H1 variant binding affinity can be performed using techniques like Biolayer Interferometry (BLI). This approach allows measurement of kon and koff rates for different H1 variants binding to recombinant nucleosome core particles .

For comparative binding studies:

• Prepare recombinant nucleosomes: Assemble octamers using recombinantly purified core histones and reconstitute nucleosome core particles with strong nucleosome positioning DNA sequence (like the "601" sequence) .

• Add linker DNA: Include a 30 bp linker at the 3' end of the positioning sequence to create a docking surface for H1 .

• Purify H1 variants: Obtain highly purified (>95% purity) H1 variants including H1.2/HIST1H1C .

• Perform binding assays: Use Biolayer Interferometry or other quantitative binding assays to determine relative affinities.

This approach enables the study of intrinsic binding preferences uncoupled from variant-specific regulation and compaction functions, as it uses defined substrates containing no histone or DNA modifications .