HIST1H1D (Ab-90) Antibody

What is HIST1H1D (Ab-90) Antibody and what protein does it target?

HIST1H1D (Ab-90) Antibody is a polyclonal antibody raised in rabbits that targets Histone H1.3, specifically around the lysine residue at position 90. The antibody recognizes the human HIST1H1D protein, which is also known by several aliases including H1 histone family member 3, H1.3, H1F3, and Histone H1c. The target protein functions as a linker histone that binds to DNA between nucleosomes, forming the macromolecular structure known as the chromatin fiber . This antibody is crucial for studying chromatin organization and epigenetic regulation in human cells.

How does HIST1H1D (H1.3) differ from other histone H1 variants in cellular distribution?

HIST1H1D (H1.3) displays a distinctive distribution pattern compared to other H1 variants. Recent super-resolution microscopy studies have revealed that H1.3, along with H1.2 and H1.5, is universally enriched at the nuclear periphery in all human cell lines analyzed and co-localizes with compacted DNA . In contrast, H1.0 shows a less pronounced peripheral localization with variability among different cell lines, while H1.4 and H1X are distributed throughout the nucleus, with H1X being enriched in high-GC regions and abundant in nucleoli . Genome-wide analyses have demonstrated that H1.3 is predominantly found in low-GC regions and B compartments of the genome, similar to H1.2, H1.5, and H1.0, forming a distinct group separate from H1.4 and H1X .

What are the validated applications for HIST1H1D (Ab-90) Antibody?

The HIST1H1D (Ab-90) Antibody has been validated for several research applications:

• ELISA (Enzyme-Linked Immunosorbent Assay): Useful for quantitative detection of the target protein

• IHC (Immunohistochemistry): For tissue section analysis with recommended dilutions of 1:10-1:100

• IF (Immunofluorescence): For cellular localization studies with recommended dilutions of 1:1-1:10

These applications make the antibody versatile for various experimental approaches in epigenetic and nuclear signaling research.

What are the recommended storage and handling conditions for maintaining antibody activity?

Upon receipt, the HIST1H1D (Ab-90) Antibody should be stored at -20°C or -80°C to maintain its activity. Repeated freeze-thaw cycles should be avoided as they may compromise antibody performance . The antibody is provided in liquid form containing 50% glycerol and 0.01M PBS (pH 7.4) with 0.03% Proclin 300 as a preservative . For short-term storage during experimental work, keeping the antibody on ice and returning it to proper freezer storage promptly after use is advisable.

How does the epitope specificity of HIST1H1D (Ab-90) Antibody compare to other HIST1H1D antibodies?

The HIST1H1D (Ab-90) Antibody recognizes a peptide sequence around lysine 90 of human Histone H1.3 . This differs from other available HIST1H1D antibodies, such as:

The distinct epitope recognition provides researchers with options for studying different functional domains of the HIST1H1D protein. The choice between these antibodies should be determined by the specific research question and whether the particular epitope region might be masked by protein-protein interactions or post-translational modifications in your experimental system.

What roles does HIST1H1D play in chromatin organization and cellular stress response?

Recent research has demonstrated that linker histones, including HIST1H1D (H1.3), are crucial for chromatin compaction and organization. While H1.0 has been specifically identified as coupling cellular mechanical behaviors to chromatin organization and being enriched in fibroblasts responding to stress , H1.3 contributes to nuclear architecture through its peripheral localization .

The enrichment of H1.3 at the nuclear periphery suggests its involvement in organizing lamina-associated domains . Depletion of specific H1 variants affects chromatin structure in variant-specific ways. For example, H1.2 knockdown triggers global chromatin decompaction , which implies that different H1 variants, including H1.3, have non-redundant roles in maintaining proper chromatin architecture and potentially in cellular responses to various stressors.

How can HIST1H1D (Ab-90) Antibody be used in combination with other techniques to study epigenetic regulation?

The HIST1H1D (Ab-90) Antibody can be effectively integrated into multi-dimensional approaches to study epigenetic regulation:

• ChIP-Seq Analysis: While this antibody hasn't been specifically validated for ChIP, researchers might optimize protocols based on similar antibodies like HIST1H1D (Ab-85) to map H1.3 genomic binding sites in relation to transcriptional activity.

• Super-Resolution Microscopy: Combining the antibody with super-resolution techniques can reveal the precise spatial organization of H1.3 within the nuclear periphery and its relationship with other chromatin components .

• Cell Stress Studies: Given the relationship between histone H1 variants and stress response , the antibody can be used to monitor changes in H1.3 distribution following mechanical stress, DNA damage, or other cellular perturbations.

• Co-Immunoprecipitation: Using the antibody for co-IP experiments can identify protein interaction partners of H1.3, providing insights into its role in chromatin remodeling complexes.

• Mass Spectrometry: Combining immunoprecipitation with mass spectrometry can identify post-translational modifications of H1.3 that may regulate its function.

What are the optimal protocols for using HIST1H1D (Ab-90) Antibody in immunohistochemistry?

For optimal IHC results with HIST1H1D (Ab-90) Antibody, consider the following protocol recommendations:

• Sample Preparation: Paraffin-embedded tissue sections should be dewaxed and hydrated properly.

• Antigen Retrieval: Based on protocols used for similar histone antibodies, high-pressure antigen retrieval in citrate buffer (pH 6.0) is recommended .

• Blocking: Block sections with 10% normal goat serum for 30 minutes at room temperature to reduce background.

• Primary Antibody Incubation: Apply HIST1H1D (Ab-90) Antibody at dilutions between 1:10-1:100 in 1% BSA and incubate overnight at 4°C .

• Detection System: For visualization, use a biotinylated secondary antibody followed by an HRP conjugated detection system .

• Controls: Include positive controls (tissues known to express HIST1H1D) and negative controls (primary antibody omitted) to validate staining specificity.

The exact protocol may require optimization based on specific tissue types and fixation methods.

What factors should be considered when validating the specificity of HIST1H1D (Ab-90) Antibody?

Validating the specificity of HIST1H1D (Ab-90) Antibody is crucial for reliable experimental results. Consider these approaches:

• Western Blotting: Confirm the antibody detects a single band at the expected molecular weight (~22 kDa) for HIST1H1D.

• Peptide Competition Assay: Pre-incubate the antibody with excess immunizing peptide to demonstrate signal reduction.

• siRNA Knockdown: Show reduced signal in cells where HIST1H1D expression has been reduced by siRNA.

• Knockout Validation: If available, test antibody reactivity in HIST1H1D knockout cells or tissues.

• Cross-Reactivity Testing: Evaluate potential cross-reactivity with other H1 variants, particularly those with high sequence homology to H1.3.

• Multiple Antibody Comparison: Compare staining patterns with other validated HIST1H1D antibodies targeting different epitopes (like Ab-85 or Ab-16) .

• Tissue Panel Testing: Validate expression patterns across tissues with known differential expression of HIST1H1D.

How should researchers interpret different nuclear distribution patterns of HIST1H1D in various cell types?

When interpreting HIST1H1D nuclear distribution patterns:

• Peripheral Enrichment: The strong enrichment of H1.3 at the nuclear periphery in all cell types examined indicates its role in organizing lamina-associated domains and heterochromatin near the nuclear envelope . This pattern is consistent across different cell lines and represents a universal feature of H1.3 distribution.

• Cell Type Variations: While the peripheral localization is consistent, subtle variations in intensity or pattern between cell types may reflect cell-specific chromatin organization or differentiation status. These differences should be quantified using image analysis software.

• Co-localization Analysis: The co-localization of H1.3 with compacted DNA suggests its role in maintaining heterochromatin. When interpreting co-localization data, consider that H1.3 belongs to a group of H1 variants (along with H1.2 and H1.5) that share similar distribution patterns and may have partially redundant functions .

• Changes Upon Cellular Perturbation: Alterations in H1.3 distribution following treatments should be interpreted in the context of chromatin reorganization. For example, shifts from peripheral to more nucleoplasmic distribution might indicate chromatin decompaction or activation of previously silenced genomic regions.

What approaches are recommended for quantifying changes in HIST1H1D localization in response to experimental treatments?

For quantitative analysis of HIST1H1D localization changes:

• Radial Distribution Analysis: Measure fluorescence intensity from the nuclear periphery to the center to quantify the degree of peripheral enrichment before and after treatments.

• Co-localization Metrics: Calculate Pearson's or Mander's coefficients to quantify co-localization with markers of heterochromatin or euchromatin.

• High-Content Imaging: Automated microscopy platforms can analyze hundreds of cells to provide statistically robust measurements of distribution changes.

• Fluorescence Recovery After Photobleaching (FRAP): If using tagged versions of H1.3, FRAP can measure mobility changes that might accompany redistribution.

• ChIP-Seq Comparison: Comparing genomic binding profiles before and after treatment can provide mechanistic insights into redistribution patterns observed microscopically.

• 3D Reconstruction: Super-resolution microscopy with 3D reconstruction can provide detailed spatial information about H1.3 redistribution that might not be apparent in 2D imaging .

How does the functional relationship between HIST1H1D and other histone variants impact experimental design?

Understanding the functional relationships between HIST1H1D and other histone variants is critical for experimental design:

• Compensatory Mechanisms: Studies have shown compensatory upregulation of other H1 variants after depletion of individual linker histones . Therefore, knockdown experiments targeting HIST1H1D should assess potential changes in other H1 variants that might mask phenotypes.

• Variant-Specific Functions: While H1.3 shares distribution patterns with H1.2 and H1.5, each variant likely has unique functions. Experimental designs should account for both shared and variant-specific roles, possibly using multiple antibodies targeting different H1 variants .

• Cell Type Selection: The relative expression levels of H1 variants differ between cell types. Interestingly, in cell lines lacking H1.3 and H1.5, H1.4 and H1.0 show a more peripheral distribution, suggesting functional compensation . Choose cell models with appropriate H1 variant expression profiles for your research question.

• Combinatorial Approaches: To fully understand H1.3 function, consider combinatorial knockdown or overexpression of multiple H1 variants to reveal redundant or synergistic effects.

• Developmental Context: The expression of H1 variants changes during differentiation, with H1.0 accumulating during differentiation . Experimental timing should account for these developmental dynamics.

What emerging technologies might enhance HIST1H1D (Ab-90) Antibody applications in epigenetic research?

Several emerging technologies promise to expand HIST1H1D antibody applications:

• Spatial Omics: Combining antibody-based detection with spatial transcriptomics or proteomics could reveal how H1.3 distribution correlates with gene expression patterns at the single-cell level.

• Live-Cell Imaging: Development of intrabodies or nanobodies derived from HIST1H1D antibodies could enable live-cell tracking of H1.3 dynamics.

• CRISPR-Cas9 Genome Editing: Precise epitope tagging of endogenous HIST1H1D locus could provide complementary approaches to antibody-based detection.

• Proximity Labeling: Using antibodies to target enzymes like TurboID or APEX2, researchers could map the local protein environment of H1.3 in different nuclear compartments.

• Single-Cell ChIP-Seq: Adapting HIST1H1D antibodies for single-cell chromatin profiling could reveal cell-to-cell variation in H1.3 genomic binding patterns.

How might HIST1H1D studies contribute to understanding disease mechanisms and potential therapeutic approaches?

Research into HIST1H1D has significant implications for disease understanding:

• Cancer Biology: Dysregulation of histone H1 variants has been implicated in cancer. Studies suggest H1.0 expression is associated with less aggressive tumor phenotypes , raising questions about the roles of other variants like H1.3 in tumorigenesis.

• Stress-Related Disorders: Given the role of histone H1 variants in stress responses , understanding H1.3 function could provide insights into cellular adaptation to various stressors.

• Developmental Disorders: The dynamic expression of H1 variants during differentiation suggests their importance in development. Abnormal H1.3 function might contribute to developmental disorders.

• Epigenetic Therapeutics: Targeting specific interactions involving H1.3 could potentially offer novel approaches to modulate chromatin states in disease contexts.

• Biomarker Development: The distribution pattern of H1.3 could serve as a biomarker for certain cellular states or disease progression, particularly in contexts where nuclear architecture is altered.

Research using HIST1H1D (Ab-90) Antibody will continue to enhance our understanding of chromatin biology and potentially reveal new therapeutic targets for diseases involving epigenetic dysregulation.