HIST1H1D (Ab-85) Antibody

What is HIST1H1D and what specific protein does the Ab-85 antibody recognize?

HIST1H1D (Ab-85) Antibody specifically recognizes histone H1.3, a linker histone involved in DNA packaging and chromatin structure. This antibody was developed using a peptide sequence around the site of lysine 85 derived from human histone H1.3. The antibody plays a crucial role in detecting this specific histone variant, which is essential for nucleosome organization and regulates gene expression through chromatin accessibility modulation . Recent research has highlighted the importance of this particular lysine residue (K85) as a target for post-translational modifications that can significantly alter chromatin dynamics and cellular function .

What are the validated applications for HIST1H1D (Ab-85) Antibody?

The HIST1H1D (Ab-85) Antibody has been validated for multiple research applications:

For immunohistochemistry applications, the antibody has been specifically validated in paraffin-embedded human cervical cancer tissues using a Leica BondTM system. The protocol involves antigen retrieval through high-pressure citrate buffer treatment (pH 6.0), blocking with 10% normal goat serum, and overnight primary antibody incubation at 4°C, followed by detection using a biotinylated secondary antibody and visualization with an HRP conjugated SP system .

How should HIST1H1D (Ab-85) Antibody be stored and handled?

For optimal performance and stability, HIST1H1D (Ab-85) Antibody should be stored in its liquid form consisting of 0.03% Proclin 300 preservative in 50% glycerol and 0.01M PBS at pH 7.4. This formulation helps maintain antibody integrity during storage and handling procedures. The antibody has been affinity-purified to ensure specificity and reduce background signals in experimental applications . Researchers should avoid repeated freeze-thaw cycles and maintain aliquots at recommended temperatures to preserve antibody activity throughout the research project duration.

How can HIST1H1D (Ab-85) Antibody be used to study histone modifications in cancer research?

HIST1H1D (Ab-85) Antibody serves as a valuable tool for investigating histone H1 modifications in cancer, particularly the crucial K85 monomethylation. This specific modification has been implicated in promoting stemness features in squamous cell carcinoma of the head and neck (SCCHN). Research demonstrates that the methyltransferase WHSC1 directly monomethylates lysine 85 in the conserved globular DNA-binding region of linker histone H1.4, influencing transcriptional activation of stemness-related genes like OCT4 .

For researchers investigating cancer stem cell biology, protocols can be designed to compare H1 modifications between normal and cancer tissues. This typically involves:

• Chromatin immunoprecipitation with HIST1H1D (Ab-85) Antibody

• Western blotting analysis of methylation status

• Correlation with stemness markers expression

• Functional validation through sphere formation assays

When conducting sphere formation assays to assess stemness properties, researchers should seed 5000 cells in low-attachment plates using serum-free medium supplemented with epidermal growth factor (20 ng/ml), allow sphere development for 7 days, and quantify results across multiple replicate wells .

What experimental approaches can be used to study the relationship between HIST1H1D modifications and gene expression?

Investigating the relationship between HIST1H1D modifications and gene expression requires integrated experimental approaches:

• ChIP-Seq Analysis: Using HIST1H1D (Ab-85) Antibody for chromatin immunoprecipitation followed by next-generation sequencing to map genome-wide distribution of H1.3 and its modified forms.

• RNA-Seq Correlation: Comparing transcriptional profiles between wild-type and mutant cells (e.g., H1.4K85A or H1.4K85R) to identify genes affected by specific histone modifications.

• RT-PCR Validation: Confirming expression changes in candidate genes using targeted primers. For example, OCT4 expression analysis requires specific primers and comparison to housekeeping genes like GAPDH .

• Functional Validation: Using siRNA knockdown of relevant methyltransferases (e.g., WHSC1) to determine causality between histone modification and gene expression changes. Transfection should be performed with appropriate controls (siNC) using reagents like Lipofectamine RNAimax at 50 nM final concentration for 72 hours before analysis .

These experimental approaches should be integrated to provide comprehensive insights into how specific histone modifications influence gene expression patterns and cellular phenotypes.

How does HIST1H1D (Ab-85) Antibody perform in chromatin immunoprecipitation experiments?

HIST1H1D (Ab-85) Antibody has demonstrated reliable performance in chromatin immunoprecipitation (ChIP) experiments, providing insights into histone H1.3 genomic distribution and its association with specific chromatin states. For optimal ChIP results, researchers should:

• Cross-link Protein-DNA Complexes: Typically using 1% formaldehyde for 10 minutes at room temperature.

• Sonication Optimization: Fragment chromatin to 200-500bp fragments, verifying size by gel electrophoresis.

• Immunoprecipitation: Use 300-800 μg of whole-cell extract with optimized antibody concentration, followed by protein G beads capture and thorough washing with TBS buffer (pH 7.6) .

• Controls: Include input controls, IgG controls, and positive controls targeting known histone marks.

• Analysis: Perform qPCR for targeted regions or proceed to sequencing for genome-wide analysis.

Studies have revealed that different H1 variants show distinct distribution patterns across the genome. While some H1 variants are depleted at transcriptionally active promoters, H1.2 (a related variant) shows differential enrichment patterns compared to other variants, being less abundant at transcription start sites of inactive genes but enriched at chromosomal domains with low GC content .

What are common technical challenges when using HIST1H1D (Ab-85) Antibody for Western blotting, and how can they be addressed?

When performing Western blotting with HIST1H1D (Ab-85) Antibody, researchers may encounter several technical challenges that require methodological adjustments:

• Cross-reactivity Issues: Due to high sequence similarity between histone variants, cross-reactivity may occur. To address this:

  • Use nuclear extracts rather than whole cell lysates to enrich for nuclear proteins

  • Include appropriate blocking agents (5% BSA often performs better than milk for histone antibodies)

  • Optimize antibody dilution within the recommended range

  • Consider using recombinant H1.3 as a positive control

• Low Signal Intensity: Histone detection may present challenges due to epitope accessibility:

  • Ensure complete protein transfer using specialized transfer conditions for small proteins

  • Extend primary antibody incubation to overnight at 4°C

  • Use enhanced chemiluminescence detection systems with appropriate exposure times

  • Consider nuclear extraction methods that preserve post-translational modifications

• High Background: To reduce non-specific binding:

  • Increase washing duration and frequency

  • Optimize secondary antibody dilution

  • Consider alternative blocking reagents

  • Verify that membranes are evenly exposed and not cropped to specific bands

How can researchers distinguish between different histone H1 variants in their experiments?

Distinguishing between highly similar histone H1 variants presents a significant challenge in experimental settings. Strategies to achieve variant-specific detection include:

• Variant-Specific Antibodies: Use antibodies like HIST1H1D (Ab-85) that target unique regions or modification sites. Validate specificity using recombinant proteins or knockout/knockdown controls.

• Expression Systems: Generate cell lines stably expressing tagged versions of specific H1 variants (e.g., FLAG-H1.4-WT versus FLAG-H1.4K85A) to distinguish between endogenous and exogenous proteins and their mutant forms .

• Mass Spectrometry: Employ proteomics approaches to identify variant-specific peptides and modifications. This technique has successfully identified H1 as a WHSC1-interacting candidate and confirmed H1K85 mono-methylation .

• CRISPR-Based Approaches: Generate cell lines with specific mutations (e.g., H1.4K85R) to study the functional impact of specific residues in particular H1 variants. Confirm mutations by Sanger sequencing before proceeding with experiments .

• ChIP-Seq Analysis: Compare genomic distribution patterns of different H1 variants, as studies have shown that variants like H1.2 have distinctive enrichment patterns compared to other H1 variants .

What is the significance of H1 histone K85 methylation in cancer research?

The methylation of histone H1 at lysine 85 has emerged as a significant epigenetic modification with implications for cancer biology, particularly in squamous cell carcinoma of the head and neck (SCCHN). Research using antibodies like HIST1H1D (Ab-85) has revealed that:

• WHSC1-Mediated Methylation: The methyltransferase WHSC1 specifically monomethylates H1.4 at K85 within the conserved globular DNA-binding region, representing a novel substrate beyond the canonical H3K36 methylation target .

• Stemness Induction: This specific methylation event promotes stemness features in SCCHN cells through transcriptional activation of stemness-related genes such as OCT4. This provides a mechanistic link between histone modifications and cancer stem cell properties .

• Therapeutic Targeting: The identification of this specific modification provides rationale for developing WHSC1 inhibitors as potential therapeutic agents for SCCHN patients. Understanding the structural and functional consequences of this modification may inform drug development strategies .

• Experimental Models: Researchers have developed valuable tools to study this modification, including:

  • CRISPR-generated H1.4K85R mutant cell lines

  • Stably transfected cells expressing FLAG-H1.4-WT versus FLAG-H1.4K85A

  • Methylation-specific antibodies for detection of the modification in vivo

The study of K85 methylation exemplifies how specific histone modifications can drive cancer-relevant biological processes and potentially serve as therapeutic targets.

How do different H1 histone variants distribute across chromatin in normal versus disease states?

Research using techniques involving antibodies like HIST1H1D (Ab-85) has revealed distinct distribution patterns of H1 histone variants in normal and disease conditions:

• Promoter Occupancy: H1 variants show differential depletion at promoters depending on transcriptional status. Notably, H1.2 shows unique distribution patterns compared to other variants, being less abundant at transcription start sites of inactive genes .

• Genomic Domain Association: Different H1 variants associate with distinct chromosomal domains. H1.2 is particularly enriched in regions characterized by low guanine-cytosine (GC) content and is associated with lamina-associated domains, which has implications for nuclear architecture regulation .

• Cancer-Specific Patterns: In breast cancer cells, different H1 variants show distinctive distribution patterns, with some variants being enriched at specific promoters that tend to be repressed. This suggests variant-specific roles in gene regulation during cancer progression .

• Methodological Approaches: To investigate these distribution patterns, researchers have employed:

  • Variant-specific antibodies to H1

  • Hemagglutinin (HA)-tagged recombinant H1 variants expressed in cancer cells

  • Genome-wide ChIP-seq approaches to map distribution patterns

  • Correlation with transcriptional status and chromatin features

Understanding these distribution patterns may reveal how alterations in histone variant localization contribute to disease states and identify potential intervention points for therapeutic development.

What emerging technologies might enhance the utility of HIST1H1D (Ab-85) Antibody in epigenetic research?

Several cutting-edge technologies are poised to expand the research applications of HIST1H1D (Ab-85) Antibody in epigenetic studies:

• CUT&RUN and CUT&Tag: These techniques offer advantages over traditional ChIP by providing higher resolution, requiring fewer cells, and reducing background. Adapting HIST1H1D (Ab-85) Antibody to these platforms could enhance mapping of H1.3 distribution and its modifications across the genome.

• Single-Cell Epigenomics: Combining HIST1H1D (Ab-85) Antibody with single-cell technologies could reveal cell-to-cell variability in H1.3 distribution and modifications, particularly relevant in heterogeneous tumor samples where stemness features may vary among subpopulations .

• Proximity Ligation Assays: These could identify proteins interacting with specifically modified H1.3, enhancing our understanding of how K85 methylation influences recruitment of transcriptional machinery or chromatin modifiers.

• CRISPR Epigenome Editing: Using dCas9 fusions to specifically modify H1.3 at specific genomic loci could provide causal evidence for the role of this histone variant in gene regulation and disease processes.

• Integrative Multi-Omics: Combining HIST1H1D antibody-based chromatin profiling with transcriptomics, proteomics, and metabolomics could provide comprehensive insights into how H1.3 modifications coordinate cellular responses in normal and disease states.

These technological advancements would significantly enhance the research utility of the HIST1H1D (Ab-85) Antibody beyond its current validated applications in ELISA, IHC, and ChIP .