HIST1H1B (Ab-48) Antibody

What is HIST1H1B and what is its cellular function?

HIST1H1B (Histone H1.5) is a linker histone that binds to DNA between nucleosomes, forming the macromolecular structure known as the chromatin fiber. Histones H1 are necessary for the condensation of nucleosome chains into higher-order structured fibers. Beyond its structural role, HIST1H1B functions as a regulator of individual gene transcription through mechanisms involving chromatin remodeling, nucleosome spacing, and DNA methylation . As a linker histone, it helps establish and maintain higher-order chromatin structure, influencing DNA accessibility and consequently gene expression patterns.

What epitope does the HIST1H1B (Ab-48) Antibody recognize?

The HIST1H1B (Ab-48) Antibody recognizes a peptide sequence surrounding the lysine residue at position 48 (Lys48) in human Histone H1.5. This antibody was developed using a synthetic peptide corresponding to this region as the immunogen . Understanding the specific epitope recognized by this antibody is crucial for interpreting experimental results, particularly when studying post-translational modifications or protein interactions that might affect epitope accessibility.

What are the validated applications for HIST1H1B (Ab-48) Antibody?

The HIST1H1B (Ab-48) Antibody has been validated for the following applications:

While these are the manufacturer-validated applications, researchers should perform their own validation when applying this antibody to new experimental systems or techniques .

How does HIST1H1B contribute to cancer progression mechanisms?

HIST1H1B has been identified as a promoter of basal-like breast cancer (BLBC) progression through multiple mechanisms. Research has demonstrated that HIST1H1B expression enhances breast cancer cell migration, invasion, and proliferation. In functional studies, overexpression of HIST1H1B in SUM159, BT549, and Hs578T cells led to increased proliferation, while knockdown in MDA-468 and BT20 cells caused a remarkable decrease in proliferation .

HIST1H1B promotes tumorigenicity both in vitro and in vivo. Soft-agar assays revealed that HIST1H1B expression increases colony formation, while xenograft models showed that knockdown of HIST1H1B expression reduced tumor growth. Clinical data analysis has found significant correlations between high HIST1H1B expression and:

• Larger tumor size

• Higher histological grade (particularly Grade 3 tumors)

• Increased probability of metastasis

• Poorer patient survival

These findings collectively suggest HIST1H1B functions as an oncogenic factor in breast cancer, potentially through epigenetic regulation of genes involved in cell proliferation, migration, and invasion.

What is the relationship between HIST1H1B and histone modifications in cancer?

Recent research has uncovered important connections between histone H1 proteins and histone modifications in cancer contexts. In squamous cell carcinoma of the head and neck (SCCHN), WHSC1 (a histone methyltransferase) has been shown to mono-methylate histone H1 at lysine 85 (H1K85), which may enhance stemness features in cancer cells . While this study specifically examined H1.4K85 mono-methylation, it suggests potential regulatory mechanisms that might be relevant to HIST1H1B and its role in cancer progression.

The relationship between HIST1H1B and other histone modifications represents an important area for further investigation, as these interactions likely contribute to the epigenetic dysregulation observed in cancer. Researchers should consider examining how HIST1H1B expression or modifications affect the broader histone modification landscape in their experimental systems.

What gene expression changes are associated with HIST1H1B overexpression in cancer?

Studies analyzing multiple gene expression datasets have established that HIST1H1B overexpression is significantly associated with basal-like breast cancer (BLBC), a subtype known for its aggressive clinical behavior. Specifically, HIST1H1B has been found to regulate the expression of CSF2 (Colony Stimulating Factor 2), as demonstrated through ChIP assays .

The observed relationship between HIST1H1B expression and cancer progression suggests it likely influences numerous genes involved in cellular proliferation, migration, invasion, and stemness. Researchers interested in the downstream effects of HIST1H1B should consider genome-wide approaches such as RNA-seq or ChIP-seq to comprehensively identify gene expression changes and direct binding targets.

What are the optimal conditions for using HIST1H1B (Ab-48) Antibody in immunohistochemistry?

For optimal immunohistochemistry (IHC) results with HIST1H1B (Ab-48) Antibody, researchers should follow these methodological guidelines:

• Tissue Preparation: Use formalin-fixed, paraffin-embedded (FFPE) human tissue sections. Optimal thickness is typically 4-6 μm.

• Antigen Retrieval: Perform heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0). Test both to determine optimal conditions for your specific tissue.

• Blocking: Block endogenous peroxidase activity with 3% hydrogen peroxide, followed by protein blocking with 5% normal serum.

• Primary Antibody Incubation: Dilute according to manufacturer's recommendations. Incubate overnight at 4°C or for 1-2 hours at room temperature. Always perform optimization with different dilutions.

• Detection System: Use an appropriate detection system compatible with rabbit primary antibodies.

• Controls: Include positive control tissues with known HIST1H1B expression and negative controls (primary antibody omitted) .

While these general guidelines provide a starting point, optimization for specific experimental contexts is essential for reliable results.

How should researchers troubleshoot non-specific binding when using HIST1H1B (Ab-48) Antibody?

When encountering non-specific binding with HIST1H1B (Ab-48) Antibody, methodically address the issue using these approaches:

• Antibody Dilution Optimization: Test a dilution series (e.g., 1:100, 1:250, 1:500, 1:1000) to identify the optimal concentration that maximizes specific signal while minimizing background.

• Blocking Protocol Enhancement: Increase blocking time or concentration. Consider using different blocking agents (BSA, normal serum, commercial blocking solutions) compatible with your detection system.

• Washing Stringency: Increase the number and duration of washing steps with PBST (PBS + 0.1% Tween-20) to remove weakly bound antibodies.

• Cross-Reactivity Assessment: Perform peptide competition assays using the immunizing peptide to confirm specificity. Signal that disappears with peptide pre-incubation indicates specific binding.

• Sample Preparation Optimization: Ensure proper fixation and antigen retrieval. Overfixation can cause high background, while inadequate fixation may lead to non-specific binding.

• Secondary Antibody Evaluation: Test different secondary antibodies or detection systems to identify potential sources of non-specific signals.

• Tissue-Specific Considerations: Some tissues have high endogenous peroxidase activity or biotin, requiring additional blocking steps specific to these issues.

Document all optimization steps systematically to develop a reproducible protocol tailored to your experimental system.

What are the recommended storage and handling procedures to maintain antibody activity?

To preserve the activity and specificity of HIST1H1B (Ab-48) Antibody, adhere to these storage and handling procedures:

• Long-term Storage: Store at -20°C or -80°C in small aliquots to avoid repeated freeze-thaw cycles, which can degrade antibody quality .

• Working Stock Handling: For short-term use (1-2 weeks), store a working dilution at 4°C.

• Freeze-Thaw Minimization: Limit freeze-thaw cycles to less than 5 times, as repeated freeze-thaw can reduce antibody activity.

• Buffer Composition: The antibody is supplied in a buffer containing 50% glycerol and 0.03% Proclin 300 in 0.01M PBS (pH 7.4) , which helps maintain stability during storage.

• Temperature Transitions: Allow frozen antibody to thaw completely at 4°C before use, avoiding rapid temperature changes.

• Contamination Prevention: Use sterile technique when handling antibody solutions to prevent microbial contamination.

• Expiration Monitoring: Record date of receipt and track usage time, as antibody performance may decline over extended storage periods.

• Transport Conditions: If transporting between facilities, maintain cold chain using dry ice or sufficient cooling packs.

Proper storage and handling significantly impact experimental reproducibility and reliability of results when using antibodies for research applications.

How can HIST1H1B (Ab-48) Antibody be used to study epigenetic regulation in cancer research?

HIST1H1B (Ab-48) Antibody serves as a valuable tool for investigating epigenetic regulation in cancer research through several methodological approaches:

• ChIP-qPCR and ChIP-seq Analysis: Utilize this antibody in chromatin immunoprecipitation assays to identify genomic regions where HIST1H1B binds. This approach can reveal its role in regulating specific genes, as demonstrated in studies examining HIST1H1B association with CSF2 in breast cancer .

• Co-Immunoprecipitation Studies: Employ the antibody to isolate HIST1H1B protein complexes from cancer cells, enabling identification of interacting proteins that may contribute to its regulatory functions.

• Immunohistochemistry of Patient Samples: Apply IHC techniques to analyze HIST1H1B expression levels across different cancer types, grades, and stages, establishing correlations with clinical outcomes as shown in breast cancer research .

• Functional Studies with Expression Modulation: Compare epigenetic landscapes in cells with normal, overexpressed, or knocked-down HIST1H1B levels using techniques like ATAC-seq or histone modification ChIP-seq to understand its impact on chromatin accessibility.

• Analysis of Post-translational Modifications: Combine with modification-specific antibodies to investigate how HIST1H1B modifications, such as methylation at specific lysine residues, correlate with cancer progression .

When designing these experiments, researchers should include appropriate controls and consider cell type-specific effects, as HIST1H1B's epigenetic functions may vary across different cancer contexts.

What are the best practices for using HIST1H1B (Ab-48) Antibody in prognostic studies of breast cancer?

For prognostic studies of breast cancer using HIST1H1B (Ab-48) Antibody, researchers should follow these methodological best practices:

• Patient Cohort Selection: Include diverse breast cancer subtypes with adequate representation of basal-like breast cancers (BLBC), where HIST1H1B has shown strongest prognostic value . Ensure comprehensive clinical data including tumor grade, size, metastasis status, and long-term survival information.

• Tissue Microarray Construction: Develop tissue microarrays with multiple cores per tumor to account for intratumoral heterogeneity, including both tumor center and invasive margins when possible.

• Staining Protocol Standardization: Establish a standardized immunohistochemistry protocol with validated positive and negative controls. Document all experimental conditions meticulously for reproducibility.

• Scoring System Development:

  • Implement a quantitative scoring system incorporating both staining intensity and percentage of positive cells

  • Consider using digital pathology and automated scoring to reduce subjective bias

  • Validate scoring through independent assessment by multiple pathologists

• Statistical Analysis Framework:

  • Categorize HIST1H1B expression using statistically rigorous cutoff determination methods

  • Perform survival analyses including Kaplan-Meier curves and Cox proportional hazards modeling

  • Conduct multivariate analyses to determine independence from established prognostic factors

• Correlation with Molecular Features: Analyze associations between HIST1H1B expression and known molecular features of breast cancer (hormone receptor status, HER2 status, proliferation markers).

• Validation Cohort Testing: Validate findings in an independent patient cohort to confirm reproducibility and clinical relevance.

Research has already demonstrated that high HIST1H1B expression correlates with larger tumor size, higher grade, increased metastatic potential, and poorer survival in breast cancer patients , establishing its potential as a prognostic biomarker.

How can researchers integrate HIST1H1B (Ab-48) Antibody studies with other histone modification analyses?

To comprehensively investigate epigenetic regulation, researchers should integrate HIST1H1B (Ab-48) Antibody studies with broader histone modification analyses using these methodological approaches:

• Sequential ChIP (Re-ChIP) Analysis: Perform chromatin immunoprecipitation first with HIST1H1B (Ab-48) Antibody, followed by a second immunoprecipitation with antibodies against specific histone modifications (e.g., H3K4me3, H3K27me3, H3K9ac). This reveals genomic regions where HIST1H1B co-occurs with particular histone marks.

• Multi-Omics Integration Strategy:

  • Combine HIST1H1B ChIP-seq with RNA-seq from the same samples

  • Integrate with ATAC-seq to assess chromatin accessibility

  • Correlate with DNA methylation profiles from techniques like WGBS or RRBS

  • Analyze alongside histone modification ChIP-seq maps

• Proximity Ligation Assays (PLA): Use PLA to visualize and quantify spatial relationships between HIST1H1B and specific histone modifications in situ within cell nuclei.

• Mass Spectrometry Analysis: Employ immunoprecipitation with HIST1H1B (Ab-48) Antibody followed by mass spectrometry to identify associated histone modifications and histone-modifying enzymes.

• Functional Perturbation Studies: Investigate how HIST1H1B knockdown or overexpression affects the global landscape of histone modifications using techniques like ChIP-seq or CUT&RUN for various histone marks.

• Correlative Analysis with Known Modifiers: Examine relationships between HIST1H1B and histone-modifying enzymes like WHSC1, which has been shown to mono-methylate histone H1 at K85 , potentially influencing stemness features in cancer cells.

This integrated approach provides a comprehensive understanding of how HIST1H1B interacts with the broader epigenetic landscape to influence gene expression and cellular phenotypes in normal and disease states.

How should researchers interpret differences in HIST1H1B expression across breast cancer molecular subtypes?

When interpreting HIST1H1B expression patterns across breast cancer molecular subtypes, researchers should consider these methodological insights:

• Subtype-Specific Expression Profile Analysis: Research has established that HIST1H1B expression is particularly elevated in basal-like breast cancer (BLBC) compared to other molecular subtypes . When analyzing expression data:

  • Compare expression across all major molecular subtypes (Luminal A, Luminal B, HER2-enriched, Basal-like, Normal-like)

  • Use established molecular classification systems (PAM50, IntClust, etc.) for consistency

  • Consider expression as both continuous and categorical variables in analyses

• Biological Context Interpretation:

  • Higher HIST1H1B expression in BLBC aligns with this subtype's generally more aggressive phenotype

  • Consider whether HIST1H1B is a driver or passenger in subtype-specific biology

  • Evaluate correlation with basal markers (CK5/6, EGFR) to confirm subtype association

• Prognostic Significance Evaluation: When observed in BLBC, high HIST1H1B expression correlates with:

  • Larger tumor size

  • Higher histological grade (especially Grade 3)

  • Increased probability of metastasis

  • Poorer patient survival

• Methodological Considerations for Expression Measurement:

  • Account for technical variation between platforms (microarray vs. RNA-seq vs. IHC)

  • Normalize data appropriately for the platform used

  • Validate findings across multiple independent datasets

  • Confirm mRNA-level findings with protein-level analyses when possible

• Multivariable Analysis Framework: Determine whether HIST1H1B expression provides independent prognostic information beyond known subtype characteristics through robust statistical modeling.

These interpretation guidelines help researchers contextualize HIST1H1B expression patterns within the complex molecular landscape of breast cancer, potentially revealing new insights into subtype-specific biology and therapeutic opportunities.

What controls should be included when measuring HIST1H1B histone modifications in experimental systems?

When investigating HIST1H1B histone modifications in experimental systems, researchers should implement these essential controls:

• Antibody Validation Controls:

  • Peptide Competition Assay: Pre-incubate HIST1H1B (Ab-48) Antibody with immunizing peptide to confirm specificity

  • Knockout/Knockdown Validation: Include HIST1H1B-depleted samples to verify antibody specificity

  • Recombinant Protein Controls: Test antibody against purified recombinant HIST1H1B with and without specific modifications

• Modification-Specific Controls:

  • Modification Enzyme Manipulation: Include samples where known HIST1H1B-modifying enzymes (e.g., WHSC1 for K85 methylation ) are overexpressed or inhibited

  • Modified vs. Unmodified Peptides: Use synthetic peptides with specific modifications as standards

• Experimental System Controls:

  • Cell Cycle Synchronization: Control for cell cycle phase, as histone modifications can vary throughout the cell cycle

  • Treatment Time Course: Include multiple time points after stimulus to capture dynamic changes

  • Dose-Response Analysis: Test multiple concentrations of modifying treatments

• Technical Controls for ChIP Experiments:

  • Input Chromatin Control: Process a portion of starting chromatin material without immunoprecipitation

  • IgG Control: Perform parallel immunoprecipitation with isotype-matched IgG

  • Housekeeping Gene Regions: Include genomic regions with stable, known modification status

  • Spike-in Controls: Add exogenous chromatin from a different species for normalization

• Data Analysis Controls:

  • Biological Replicates: Minimum of three independent biological replicates

  • Technical Replicates: Multiple technical replicates for each biological sample

  • Normalization Standards: Include consistent reference genes or regions for normalization

These comprehensive controls ensure reliable detection and interpretation of HIST1H1B modifications in experimental systems, particularly important when investigating novel modifications like K85 methylation that may impact cancer cell stemness features .

How does HIST1H1B expression data correlate with patient outcomes in clinical datasets?

Analysis of HIST1H1B expression in clinical datasets reveals significant correlations with patient outcomes through multiple methodological dimensions:

• Survival Analysis Findings:

  • Kaplan-Meier survival analysis in both mRNA (NKI295 dataset) and protein level (Tang's dataset) studies demonstrates that patients with high HIST1H1B expression have significantly shorter survival compared to those with low expression

  • This survival disadvantage persists across different breast cancer cohorts, suggesting a robust prognostic role

• Tumor Characteristics Correlations:

  • Tumor Size: Clinical data analysis shows a significant association between high HIST1H1B expression and larger primary tumor size

  • Histological Grade: HIST1H1B expression is predominantly elevated in high-grade tumors, particularly Grade 3, as demonstrated in NKI295 and GSE22358 datasets

  • Metastatic Potential: Patients with high HIST1H1B expression show a significantly higher probability of metastasis in the NKI295 dataset

• Multivariate Analysis Context:

  • The prognostic value of HIST1H1B expression appears particularly strong in the context of basal-like breast cancer

  • Statistical models suggest HIST1H1B may provide additional prognostic information beyond standard clinical parameters

• Data Interpretation Framework:

  • The consistent association across multiple independent datasets strengthens the validity of HIST1H1B as a prognostic biomarker

  • The correlation with multiple aggressive features (tumor size, grade, metastasis) suggests HIST1H1B may be functionally involved in disease progression rather than merely serving as a passive biomarker

• Potential Clinical Implications:

  • Given the strong association with aggressive disease features and poor outcomes, HIST1H1B represents both a potential prognostic biomarker and therapeutic target, particularly for basal-like breast cancer patients

  • The consistency of these findings across different study cohorts supports potential clinical translation of HIST1H1B assessment into prognostic algorithms