HIST1H1D (Ab-85) Antibody

What is HIST1H1D (Ab-85) Antibody and what specific target does it recognize?

HIST1H1D (Ab-85) Antibody (PACO60611) is a polyclonal antibody produced in rabbits that specifically recognizes a peptide sequence around the lysine 85 (K85) site of human Histone H1.3. This antibody targets a crucial region within the conserved globular DNA-binding domain of the linker histone H1 . The specificity for the K85 region is particularly significant as this site undergoes post-translational modifications, including mono-methylation, which has been implicated in chromatin remodeling and gene expression regulation .

How does HIST1H1D differ from other H1 histone variants?

HIST1H1D (coding for histone H1.3) is one of seven somatic linker histone H1 variants (H1.1 to H1.5, H1.0, and H1X) present in human cells. Unlike other variants such as H1.2, H1.3 shows distinct genomic distribution patterns and is ubiquitously expressed across most cell types . Genome-wide distribution studies indicate that H1.3 (HIST1H1D) has different enrichment patterns at promoter regions compared to other variants like H1.2, which tends to be less abundant at transcription start sites of inactive genes . Additionally, the specific post-translational modifications occurring at the K85 site of H1.3 distinguish it functionally from other H1 variants in terms of its role in chromatin organization and gene regulation .

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

The HIST1H1D (Ab-85) Antibody has been validated for multiple research applications including Enzyme-Linked Immunosorbent Assay (ELISA), Immunohistochemistry (IHC), and Chromatin Immunoprecipitation (ChIP) . For ELISA applications, the recommended dilution range is 1:2000-1:10000, while for IHC applications, dilutions between 1:10-1:100 are recommended for optimal results . The antibody has demonstrated high specificity in detecting human histone H1.3, making it valuable for investigating histone modifications in various experimental contexts, particularly in studies related to chromatin structure and gene expression regulation .

How should nuclear extracts be prepared for optimal detection of HIST1H1D using Western blotting?

For optimal detection of HIST1H1D in Western blotting, nuclear extracts should be prepared using specialized nuclear extraction kits (e.g., Active Motif). The protocol should include the following steps: (1) Harvest cells and wash with PBS; (2) Lyse cells using CelLytic M cell lysis reagent containing complete protease inhibitor cocktail to prevent protein degradation; (3) Isolate nuclear fraction following the nuclear extraction kit protocol; (4) Quantify protein concentration; (5) Separate proteins by SDS-PAGE and transfer to nitrocellulose membrane; (6) Block the membrane and incubate with HIST1H1D (Ab-85) Antibody at appropriate dilution; (7) Visualize using HRP-conjugated secondary antibodies and enhanced chemiluminescence detection . It's crucial to include controls and ensure even exposure across the membrane to accurately assess histone H1.3 levels and modifications .

What are the recommended protocols for using HIST1H1D (Ab-85) Antibody in immunohistochemistry?

For immunohistochemistry applications with HIST1H1D (Ab-85) Antibody, the following protocol has been validated: (1) Deparaffinize and rehydrate tissue sections; (2) Perform antigen retrieval using high-pressure treatment in citrate buffer (pH 6.0); (3) Block sections with 10% normal goat serum for 30 minutes at room temperature; (4) Incubate with primary antibody (HIST1H1D Ab-85) diluted at 1:10-1:100 (optimal at 1:20 for paraffin-embedded samples) in 1% BSA overnight at 4°C; (5) Wash and incubate with biotinylated secondary antibody; (6) Visualize using an HRP-conjugated SP system . This protocol has been successfully employed to detect H1.3 in human cervical cancer tissues using a Leica BondTM system, demonstrating the antibody's effectiveness in analyzing histone H1.3 distribution in pathological samples .

What controls should be included when performing ChIP assays with HIST1H1D (Ab-85) Antibody?

When conducting Chromatin Immunoprecipitation (ChIP) assays with HIST1H1D (Ab-85) Antibody, the following controls are essential: (1) Input control - a small portion of chromatin prior to immunoprecipitation to normalize ChIP data; (2) Positive control - using an antibody against a histone mark known to be present in your sample (e.g., H3K4me3 for active promoters); (3) Negative control - using non-immune IgG from the same species as the primary antibody (rabbit IgG); (4) Positive locus control - primers targeting regions known to contain H1.3; (5) Negative locus control - primers targeting regions known to lack H1.3 . Additionally, when investigating interactions between HIST1H1D and other proteins or histone marks, sequential ChIP (re-ChIP) may be necessary to confirm co-occupancy at specific genomic loci .

What are common issues affecting HIST1H1D (Ab-85) Antibody specificity, and how can they be addressed?

Several factors can affect HIST1H1D (Ab-85) Antibody specificity: (1) Cross-reactivity with other H1 variants - due to high sequence similarity between histone H1 family members, conduct pre-absorption tests with recombinant histones or peptide competition assays to verify specificity; (2) Interference from post-translational modifications - the K85 region undergoes various modifications including methylation and acetylation that may affect antibody binding, so validate using modification-specific controls; (3) Fixation artifacts - overfixation can mask epitopes, so optimize fixation conditions; (4) Storage degradation - improper storage can reduce antibody specificity, so aliquot and store at -20°C with 50% glycerol as specified in the storage buffer (0.03% Proclin 300, 50% Glycerol, 0.01M PBS, pH 7.4) . When specificity issues persist, validation using recombinant H1.3 proteins with known modification states can help confirm antibody performance across applications .

How can researchers optimize ChIP protocols specifically for HIST1H1D detection?

Optimizing ChIP protocols for HIST1H1D detection requires several specific considerations: (1) Crosslinking optimization - histone H1 has dynamic chromatin binding properties, so test different formaldehyde concentrations (0.5-2%) and crosslinking times (5-15 minutes); (2) Sonication conditions - adjust sonication parameters to obtain chromatin fragments of 200-500bp, as improper fragmentation can affect H1.3 detection; (3) Antibody concentration - titrate HIST1H1D (Ab-85) Antibody to determine optimal concentration for immunoprecipitation; (4) Washing stringency - optimize salt concentrations in wash buffers to reduce background while maintaining specific signals; (5) Elution conditions - test different elution buffers to maximize recovery while preserving antibody integrity . Additionally, consider using a dual crosslinking approach with both formaldehyde and protein-protein crosslinkers (like DSG) to better capture the dynamic interactions of linker histones with chromatin .

What strategies can improve signal detection when working with low abundance H1.3 modifications?

When investigating low abundance H1.3 modifications such as K85 methylation, several strategies can enhance detection sensitivity: (1) Increase starting material - use larger cell numbers for extraction to enhance rare modification signals; (2) Employ signal amplification techniques - use tyramide signal amplification for immunohistochemistry or biotinylated secondary antibodies with streptavidin-HRP for Western blotting; (3) Optimize antibody incubation conditions - extend incubation times (overnight at 4°C) and use optimized blocking buffers to reduce background; (4) Enrich target population - fractionate chromatin or use sequential immunoprecipitation to concentrate modified H1.3; (5) Consider mass spectrometry validation - complement antibody-based detection with MS analysis of H1.3 modifications to confirm specificity . Additionally, utilizing highly sensitive detection systems such as enhanced chemiluminescence plus (ECL+) or fluorescently-labeled secondary antibodies can significantly improve detection of low abundance H1.3 modifications .

How can HIST1H1D (Ab-85) Antibody be used to investigate the role of H1.3K85 methylation in cancer progression?

To investigate H1.3K85 methylation in cancer progression, researchers can implement a multi-faceted approach: (1) Compare H1.3K85 methylation levels across normal, premalignant, and malignant tissues using immunohistochemistry with HIST1H1D (Ab-85) Antibody; (2) Conduct ChIP-seq analysis to map genome-wide distribution of methylated H1.3K85 in cancer versus normal cells, identifying differential binding patterns; (3) Correlate H1.3K85 methylation with expression of known oncogenes or tumor suppressors using RT-qPCR following ChIP; (4) Employ CRISPR-Cas9 to generate H1.3K85 mutants (K85R or K85A) to assess functional consequences of preventing this modification; (5) Investigate the relationship between H1.3K85 methylation and methyltransferases like WHSC1, which has been shown to monomethylate H1 at K85 and induce stemness features in squamous cell carcinoma of the head and neck (SCCHN) . This approach would provide insights into how H1.3K85 methylation contributes to chromatin dynamics and gene expression changes during cancer development and progression .

What approaches can be used to distinguish between different post-translational modifications at the K85 site of H1.3?

Distinguishing between different post-translational modifications at the K85 site of H1.3 requires specialized techniques: (1) Modification-specific antibodies - use antibodies that specifically recognize mono-, di-, or tri-methylation, acetylation, or other modifications at K85; (2) Mass spectrometry - employ high-resolution MS/MS approaches with electron transfer dissociation (ETD) or electron capture dissociation (ECD) to precisely identify and quantify K85 modifications; (3) In vitro methylation assays - conduct assays using purified H1.3 and specific methyltransferases like WHSC1, followed by Western blotting with modification-specific antibodies; (4) 2D gel electrophoresis - separate H1.3 isoforms based on charge and mass differences resulting from different modifications; (5) Sequential ChIP - perform consecutive immunoprecipitations with antibodies against different modifications to identify co-occurrence patterns . These approaches are crucial for understanding the complex "histone code" of H1.3 and how different modifications at K85 might lead to distinct functional outcomes in gene regulation and chromatin organization .

How can researchers investigate the relationship between HIST1H1D and other histone variants in chromatin organization?

To investigate relationships between HIST1H1D (H1.3) and other histone variants in chromatin organization, researchers should consider: (1) Co-immunoprecipitation assays using HIST1H1D (Ab-85) Antibody to identify proteins interacting with H1.3, followed by mass spectrometry for unbiased identification of binding partners; (2) Sequential ChIP (re-ChIP) combining HIST1H1D (Ab-85) Antibody with antibodies against other histone variants to identify regions of co-occupancy; (3) Genome-wide mapping techniques such as ChIP-seq to compare distribution patterns of H1.3 with other H1 variants (particularly H1.2, which shows distinctive genomic localization); (4) CRISPR-mediated knockout or knockdown of HIST1H1D followed by ChIP-seq for other histone variants to assess compensatory or consequential changes in their distribution; (5) Fluorescence Recovery After Photobleaching (FRAP) experiments with fluorescently tagged H1 variants to compare their dynamics and mobility in the context of different chromatin states . These approaches would help elucidate how the differential distribution of H1.3 relative to other variants (such as H1.2, which is depleted at transcription start sites of inactive genes) contributes to higher-order chromatin structure and gene regulation .

How does H1.3K85 methylation integrate with other epigenetic marks in regulating gene expression?

H1.3K85 methylation operates within a complex network of epigenetic modifications that collectively regulate gene expression. To investigate these relationships: (1) Perform ChIP-seq with HIST1H1D (Ab-85) Antibody alongside antibodies for core histone modifications (H3K4me3, H3K27me3, H3K9me3, etc.) to map co-occurrence patterns; (2) Employ bioinformatic approaches to correlate H1.3K85 methylation with DNA methylation profiles, identifying potential synergistic or antagonistic relationships; (3) Conduct sequential ChIP experiments to determine if H1.3K85 methylation co-exists with specific core histone modifications at particular genomic loci; (4) Investigate the temporal dynamics of H1.3K85 methylation during cellular processes like differentiation or oncogenic transformation relative to other epigenetic changes; (5) Analyze the effects of methyltransferase inhibitors on both H1.3K85 methylation and other histone modifications to uncover regulatory hierarchies . Research indicates that WHSC1-mediated H1.3K85 mono-methylation may influence stemness features in cancer cells, suggesting it plays a role in transcriptional activation of pluripotency genes like OCT4, potentially working in concert with or opposition to other epigenetic marks .

What role does HIST1H1D play in B-cell mediated immunity and antibody production?

The role of HIST1H1D in B-cell mediated immunity can be investigated through several approaches: (1) Analyze H1.3 expression and distribution during B-cell activation and differentiation using HIST1H1D (Ab-85) Antibody in flow cytometry and ChIP-seq; (2) Compare H1.3K85 methylation patterns in naive versus activated B cells to identify potential regulatory mechanisms during immune response; (3) Perform knockout or knockdown of HIST1H1D in B-cell models followed by assessment of immunoglobulin class switching, proliferation, and antibody production; (4) Investigate potential interactions between H1.3 and BAP1 (BRCA1-associated protein 1), which has been shown to play a crucial role in B-cell intrinsic regulation of antibody-mediated immunity through histone modifications; (5) Conduct co-immunoprecipitation experiments to identify B-cell specific interaction partners of H1.3 that may mediate its function in humoral immunity . Recent studies have shown that epigenetic regulation through histone modifications is central to B-cell activation and antibody-mediated immune responses, making H1.3 and its modifications potentially important players in this process .

What are the implications of HIST1H1D distribution patterns for understanding chromatin accessibility and nuclear organization?

The distribution patterns of HIST1H1D (H1.3) have significant implications for understanding chromatin accessibility and nuclear organization: (1) Compare H1.3 distribution using HIST1H1D (Ab-85) Antibody in ChIP-seq with accessibility maps generated by ATAC-seq or DNase-seq to identify correlations between H1.3 binding and chromatin compaction; (2) Investigate H1.3 enrichment in relation to lamina-associated domains (LADs) and topologically associated domains (TADs) to understand its role in higher-order chromatin organization; (3) Analyze the relationship between H1.3 distribution and GC content across chromosomal domains, as certain H1 variants show preferential binding to regions with specific GC compositions; (4) Employ super-resolution microscopy with HIST1H1D (Ab-85) Antibody to visualize the spatial distribution of H1.3 within the nucleus and its relationship to chromatin compartments; (5) Conduct Hi-C experiments in conjunction with H1.3 ChIP-seq to correlate H1.3 binding with three-dimensional chromatin interactions . Research indicates that different H1 variants, including H1.3, have distinct genomic distributions, with some variants like H1.2 being enriched in chromosomal domains with low GC content and associated with lamina-associated domains, suggesting specialized roles in organizing nuclear architecture .

What are the best storage and handling practices for maintaining HIST1H1D (Ab-85) Antibody activity?

To maintain optimal HIST1H1D (Ab-85) Antibody activity, follow these evidence-based storage and handling practices: (1) Store the antibody at -20°C in its recommended buffer (0.03% Proclin 300, 50% Glycerol, 0.01M PBS, pH 7.4) to preserve activity; (2) Divide the stock solution into small working aliquots to avoid repeated freeze-thaw cycles, which can significantly reduce antibody functionality; (3) When thawing aliquots, allow them to equilibrate to room temperature gradually before opening to prevent condensation that could introduce contaminants; (4) During handling, use sterile pipette tips and microcentrifuge tubes to prevent bacterial contamination; (5) For short-term storage (1-2 weeks), antibody can be kept at 4°C, but longer storage requires freezing at -20°C; (6) Monitor antibody performance regularly using positive controls, as degradation may occur over time even with optimal storage conditions . These practices help maintain the antibody's specificity and sensitivity for detecting histone H1.3 across various experimental applications.

What dilution optimization strategies should be employed for different experimental applications?

Optimizing HIST1H1D (Ab-85) Antibody dilutions for different applications requires systematic approach: (1) For ELISA applications, start with a broad dilution range (1:2000-1:10000) as recommended, then perform a dilution series within this range to identify the optimal concentration that maximizes specific signal while minimizing background; (2) For IHC applications, begin with the recommended 1:10-1:100 range, testing several dilutions on positive control tissues with known H1.3 expression levels; (3) For Western blotting, conduct an antibody titration experiment starting at 1:1000 and adjust based on signal-to-noise ratio; (4) For ChIP applications, optimize antibody concentration relative to chromatin amount, typically starting with 2-5 μg antibody per 25-100 μg chromatin; (5) Consider cell or tissue-specific factors that might affect optimal dilution, such as fixation methods, protein expression levels, and post-translational modifications . A systematic titration approach with appropriate positive and negative controls is essential for each application to ensure reliable, reproducible results while conserving valuable antibody resources.