HIST1H1B (Ab-17) Antibody

What is HIST1H1B and why is it important in epigenetic research?

HIST1H1B (also known as H1.4) is a member of the linker histone family that plays a crucial role in organizing and condensing DNA into chromatin. It is involved in regulating gene expression, DNA replication, and DNA repair processes . Recent research has demonstrated that HIST1H1B can function as either a positive or negative regulator of gene expression in tumors, making it an important target in epigenetic and cancer research . Rather than simply labeling histones as general structural proteins, researchers should approach HIST1H1B as a specific regulator that can actively modulate gene expression through direct binding to promoter regions, as demonstrated in the case of CSF2 regulation .

What are the validated applications for HIST1H1B (Ab-17) Antibody?

The HIST1H1B (Ab-17) Antibody (PACO56607) has been validated for several research applications including:

• ELISA (recommended dilution: 1:2000-1:10000)

• Immunohistochemistry (IHC) (recommended dilution: 1:20-1:200)

• Western blotting (validated but specific dilution recommendations not provided)

For optimal results in each application, researchers should perform antibody titration experiments with positive and negative controls to determine the ideal concentration for their specific experimental conditions and sample types.

How specific is the HIST1H1B (Ab-17) Antibody for human samples?

This rabbit polyclonal antibody demonstrates high reactivity with human samples and has been specifically designed against a peptide sequence around site of Ser (17) derived from Human Histone H1.5 . While the antibody's primary validation has been in human samples, cross-reactivity testing with other species should be performed if applying this antibody to non-human models. Researchers working with model organisms should conduct preliminary validation experiments to confirm reactivity before proceeding with full-scale studies.

How can HIST1H1B (Ab-17) Antibody be optimized for chromatin immunoprecipitation (ChIP) assays?

While the product documentation doesn't specifically mention ChIP applications, the demonstrated ability of HIST1H1B to bind to gene promoters (such as CSF2) suggests potential utility in ChIP assays . For ChIP optimization:

• Cross-linking optimization: Test different formaldehyde concentrations (0.5-1.5%) and incubation times (5-15 minutes) to preserve HIST1H1B-DNA interactions without overfixing.

• Chromatin fragmentation: Aim for fragments between 200-500bp for optimal resolution.

• Antibody amount: Begin with 2-5 μg antibody per ChIP reaction and adjust based on preliminary results.

• Include appropriate controls: IgG negative control and a positive control targeting a known HIST1H1B binding site such as the CSF2 promoter region.

The direct binding of HIST1H1B to the CSF2 promoter was successfully demonstrated using ChIP assay in SUM159 cells, providing a methodological blueprint for similar experiments .

What methodologies best capture HIST1H1B expression variations across different breast cancer subtypes?

Research indicates HIST1H1B expression is significantly higher in basal-like breast cancer (BLBC) compared to other subtypes . To accurately assess these variations:

• Multi-level analysis approach:

  • mRNA expression: Quantitative real-time PCR validated with multiple reference genes

  • Protein expression: Western blotting and IHC with HIST1H1B (Ab-17) Antibody

  • Genomic analysis: Copy number variation (CNV) assessment

  • Epigenetic analysis: Promoter methylation status evaluation

• Reference data comparison:

  • Analyze results against established datasets (TCGA, NKI295, GSE22358) that have shown HIST1H1B upregulation in BLBC

  • Include diverse breast cancer cell lines representing different subtypes (the study used 3 luminal and 7 BLBC cell lines for comparison)

How do post-translational modifications affect HIST1H1B detection and function?

When studying HIST1H1B, researchers should consider its post-translational modifications (PTMs), which may affect epitope recognition by the antibody and protein function. The HIST1H1B (Ab-17) Antibody specifically targets a peptide sequence around Ser (17) , making it potentially sensitive to phosphorylation at this site. Consider these methodological approaches:

• Perform parallel detection with phosphorylation-specific and total HIST1H1B antibodies

• Use phosphatase treatments on a subset of samples to determine if PTMs affect antibody recognition

• Employ mass spectrometry to identify and characterize PTMs on HIST1H1B in your experimental system

• Design functional studies to determine if PTMs alter HIST1H1B's ability to bind the CSF2 promoter and regulate gene expression

What is the optimal protocol for using HIST1H1B (Ab-17) Antibody in immunohistochemistry?

For immunohistochemistry applications, follow these methodological guidelines:

• Sample preparation:

  • Use formalin-fixed, paraffin-embedded (FFPE) tissues sectioned at 4-5 μm

  • Perform heat-induced epitope retrieval (HIER) with citrate buffer (pH 6.0)

• Staining protocol:

  • Begin with a 1:20 dilution for high-sensitivity detection and optimize as needed (range: 1:20-1:200)

  • Incubate at 4°C overnight for optimal binding

  • Use a polymer-based detection system for enhanced sensitivity

  • Include positive controls (BLBC samples) and negative controls (omit primary antibody)

• Scoring system:

  • Develop a semi-quantitative scoring system that accounts for both staining intensity and percentage of positive cells

  • Consider digital pathology tools for objective quantification

How should researchers troubleshoot non-specific binding issues with HIST1H1B (Ab-17) Antibody?

Non-specific binding can complicate HIST1H1B detection. Address this methodologically through:

• Blocking optimization:

  • Test different blocking agents (BSA, normal serum, commercial blockers)

  • Extend blocking time to 1-2 hours at room temperature

• Antibody dilution series:

  • Perform a systematic titration (e.g., 1:20, 1:50, 1:100, 1:200)

  • Determine the optimal signal-to-noise ratio for your specific samples

• Cross-reactivity testing:

  • Validate specificity using HIST1H1B knockdown samples as negative controls

  • Perform peptide competition assays with the immunizing peptide

• Sample-specific adjustments:

  • For tissues with high endogenous peroxidase activity, extend the quenching step

  • For tissues with high background, include additional washing steps with 0.1% Triton X-100

How can researchers quantitatively assess HIST1H1B expression in relation to clinical outcomes?

Based on published research, HIST1H1B expression correlates with several clinicopathological parameters in breast cancer . To quantitatively assess this relationship:

• Expression quantification methods:

  • For IHC: Use H-score or Allred scoring systems

  • For Western blotting: Normalize HIST1H1B signal to appropriate loading controls (β-actin, GAPDH)

  • For qRT-PCR: Employ the 2^-ΔΔCt method with validated reference genes

• Statistical approaches:

  • Categorical analysis: Stratify patients into high vs. low HIST1H1B expression groups using median or quartile cutoffs

  • Continuous analysis: Use HIST1H1B expression as a continuous variable in regression models

  • Survival analysis: Apply Kaplan-Meier analysis with log-rank test as demonstrated in the NKI295 dataset

• Integrated analysis:

  • Correlate HIST1H1B protein expression with mRNA levels

  • Analyze HIST1H1B expression in relation to tumor size, grade, and metastatic status

  • Perform multivariate analysis to determine independent prognostic value

Research has shown that high HIST1H1B expression correlates with larger tumor size, higher tumor grade (especially Grade 3), increased probability of metastasis, and poor survival .

How should researchers interpret variations between HIST1H1B genomic, transcriptomic, and proteomic data?

Researchers often encounter discrepancies between different molecular analyses of HIST1H1B. Use these methodological approaches to reconcile such variations:

• Integrated analysis workflow:

  • Assess HIST1H1B copy number variations (CNVs)

  • Measure promoter methylation status

  • Quantify mRNA expression

  • Determine protein levels using the HIST1H1B (Ab-17) Antibody

• Reconciliation strategies:

  • For discrepancies between copy number and expression, examine promoter methylation status, as hypomethylation can increase expression even without copy number changes

  • For discrepancies between mRNA and protein levels, investigate post-transcriptional regulation and protein stability

  • Consider technical limitations of each platform and normalize appropriately

• Validation approaches:

  • Use multiple methodologies for critical findings

  • Compare results across different patient cohorts or cell line models

  • Integrate with functional data (e.g., CSF2 expression levels)

How can researchers effectively use HIST1H1B (Ab-17) Antibody to study its role in regulating CSF2 expression?

HIST1H1B has been shown to upregulate colony-stimulating factor 2 (CSF2) expression by binding to its promoter . To investigate this regulatory mechanism:

• Chromatin binding studies:

  • Perform ChIP assays focusing on the CSF2 promoter region

  • Use sequential ChIP (re-ChIP) to identify co-binding factors

• Expression correlation analysis:

  • Assess CSF2 levels after HIST1H1B knockdown or overexpression

  • Use the HIST1H1B (Ab-17) Antibody to confirm protein levels in these models

• Functional validation approaches:

  • Reporter assays with wild-type and mutated CSF2 promoter constructs

  • CRISPR-based genome editing of HIST1H1B binding sites in the CSF2 promoter

• Downstream signaling studies:

  • Evaluate the impact on CSF2-dependent pathways

  • Measure phenotypic changes (proliferation, migration, invasion) as described in the research

What protocols are recommended for using HIST1H1B (Ab-17) Antibody in breast cancer tumorigenicity studies?

Based on published research, HIST1H1B expression promotes tumorigenicity in breast cancer models . To use the antibody in such studies:

• In vitro experimental setup:

  • Establish stable cell lines with HIST1H1B overexpression or knockdown

  • Perform proliferation assays (doubling time, MTT, BrdU incorporation)

  • Conduct soft-agar colony formation assays

  • Use mammosphere formation assays to assess cancer stem cell properties

• In vivo model development:

  • Use SCID mice for xenograft studies as described in the research

  • Inject 5×10^6 cells with altered HIST1H1B expression

  • Monitor tumor growth every 2-4 days for approximately 30 days

  • Use the antibody for tumor tissue analysis post-excision

• Antibody application in tumor analysis:

  • Perform IHC on tumor sections to confirm HIST1H1B expression status

  • Correlate HIST1H1B levels with tumor weight and volume

  • Assess CSF2 expression in the same sections

• Translational correlation:

  • Validate findings using patient-derived xenograft (PDX) models

  • Compare results with clinical data on tumor size, grade, and HIST1H1B expression in patients