Histone H1.0 Recombinant Monoclonal Antibody

What is Histone H1.0 and what distinguishes it from other histone variants?

Histone H1.0 (also known as H1F0 or H1(0)) is a member of the linker histone family that interacts with DNA between nucleosomes and mediates compaction into higher-order chromatin structures. Unlike other H1 variants, H1.0 is predominantly found in cells that are in terminal stages of differentiation or have low rates of cell division .

Research has demonstrated that H1.0 is the predominant linker histone variant expressed in fibroblasts regardless of tissue of origin . This contrasts with the traditional view that linker histones are merely structural components, as H1.0 appears to have specific regulatory functions for subsets of genes rather than acting as a general transcriptional repressor .

The functional distinctions of H1.0 include:

• Higher expression in fibroblasts compared to other H1 variants

• Important role in sensing extracellular tension and responding to organ-level stress

• Involvement in specific protein-protein interaction networks in the nucleolus

• Regulation of mechanical behaviors in activated fibroblasts

What epitopes of Histone H1.0 are typically targeted by recombinant monoclonal antibodies?

Recombinant monoclonal antibodies against Histone H1.0 typically target specific epitopes within the protein structure. Research characterizing these antibodies has identified several significant epitope regions:

• The C-terminal region: Many commercial antibodies, such as clone 2D17 ZooMAb® Rabbit recombinant monoclonal antibody, target an epitope within 18 amino acids from the C-terminal region of Histone H1.0 .

• The globular domain: Studies have shown that some monoclonal antibodies react with the globular part of H1.0. More advanced characterization has determined that these epitopes can be further subdivided into two groups :

  • Residues 24-27: Antibodies binding to this region are highly specific for H1.0

  • Residues 27-30: Antibodies targeting this region may cross-react with H5 and GH5

• Conformational epitopes: Some monoclonal antibodies react with the entire histone H1.0 but fail to react with any peptide fragments, suggesting they recognize three-dimensional conformational antigenic determinants rather than linear sequences .

Some antibodies use synthetic peptides corresponding to sequences within amino acids 1-100 of human Histone H1.0 (P07305) as immunogens .

What are the validated applications for Histone H1.0 recombinant monoclonal antibodies?

Histone H1.0 recombinant monoclonal antibodies have been validated for multiple research applications through rigorous quality control testing. Based on the available data, the following applications have been confirmed with specific methodological parameters:

For optimal results, researchers should note that actual working dilutions may need to be determined empirically as experimental conditions may vary .

How does Histone H1.0 expression correlate with cellular mechanical behaviors?

Recent research has established a significant relationship between Histone H1.0 expression and cellular mechanical behaviors, particularly in fibroblasts. Experimental evidence indicates that H1.0 couples cellular mechanical behaviors to chromatin dynamics:

• Mechanosensing function: Fibroblasts use Histone H1.0 to sense extracellular tension and respond to organ-level stress. Research shows that H1.0 is the predominant linker histone variant in fibroblasts from various injured murine tissues .

• Impact on cellular force generation: Traction force assays, where cells are seeded onto fluorescently labeled BSA beads, have demonstrated that H1.0 depletion affects the force generated by individual fibroblasts. This suggests H1.0 plays a critical role in mechanotransduction pathways .

• Tissue-specific mechanical behaviors: The relationship between H1.0 and mechanical behaviors appears to be universal across various tissues and species. Depletion of H1.0 prevented activation of fibroblasts from mouse lung, mouse skin, and human skin .

• Correlation with pathological conditions: In cardiac tissue, H1.0 levels strongly associate with metrics of heart muscle pathology and dysfunction, including left ventricular mass and echocardiography parameters (E and A amplitude) that measure the heart's ability to relax during diastole .

• Cell migration and proliferation: Experimental data shows that while H1.0 depletion does not impair cell viability, it prevents cardiac fibroblast proliferation in response to TGF-β and impairs cell migration in wound closure assays .

These findings collectively demonstrate that H1.0 functions beyond mere structural roles in chromatin and actively participates in translating mechanical cues into cellular responses.

What protein interaction networks does Histone H1.0 participate in within the nucleolus?

Histone H1.0 engages in an extensive network of protein-protein interactions within the nucleolus, suggesting functional roles beyond chromatin compaction. Pull-down experiments and proteomic analyses have revealed the following interaction networks:

• Splicing factor interactions: H1.0 has been shown to pull down 33 different splicing factors, indicating a potential role in RNA processing mechanisms .

• rRNA biogenesis: H1.0 interacts with numerous proteins involved in ribosomal RNA biogenesis, suggesting a role in regulating ribosome production and function .

• Nucleolar localization: Among the proteins identified in H1.0 pull-down experiments, 94 are found in the nucleolus, highlighting the nucleolus as a significant site of H1.0 function .

• Validated protein interactions: Quantitative binding experiments with pure recombinant proteins have confirmed specific interactions between H1.0 and several key proteins:

  • U2AF65: A splicing factor essential for 3' splice site recognition

  • SF2/ASF: A serine/arginine-rich splicing factor involved in alternative splicing

  • FACT complex: Facilitates chromatin transcription by RNA polymerase II

These interactions suggest that H1.0 functions as a central component in RNA metabolism pathways within the nucleolus, potentially linking chromatin structure to RNA processing and ribosome biogenesis.

How do researchers reconcile contradictory findings regarding Histone H1.0's effects on chromatin structure?

Researchers investigating Histone H1.0's effects on chromatin structure have encountered seemingly contradictory experimental results. These contradictions provide insight into the complex and context-dependent nature of H1.0 function:

By carefully considering these factors and designing experiments that account for the complex interaction network of H1.0, researchers can better understand its true functional impact on chromatin structure.

What are the optimal conditions for using Histone H1.0 recombinant monoclonal antibodies in immunohistochemistry?

Successful immunohistochemistry (IHC) with Histone H1.0 recombinant monoclonal antibodies requires careful optimization of several experimental parameters:

How should researchers validate the specificity of Histone H1.0 antibodies in their experimental systems?

Validating antibody specificity is critical for ensuring reliable experimental results. For Histone H1.0 recombinant monoclonal antibodies, a comprehensive validation strategy should include:

• Peptide blocking experiments: Synthetic peptide corresponding to the epitope recognized by the antibody can be used to block antibody binding. Comparing staining patterns between blocked and unblocked antibody helps identify specific versus non-specific signals. Since many H1.0 antibodies target known epitopes (e.g., residues 24-27 or 27-30 in the globular domain), corresponding synthetic peptides can be used .

• Genetic knockdown/knockout controls: Using siRNA, shRNA, or CRISPR-Cas9 to reduce or eliminate H1.0 expression provides a critical negative control. Research has demonstrated that H1.0 depletion affects fibroblast activation, proliferation, and migration, making these phenotypes useful readouts for validation .

• Cross-reactivity assessment: Testing the antibody against recombinant proteins of other H1 variants (especially those with similar epitope regions) helps determine specificity. Some antibodies targeting residues 27-30 have shown cross-reactivity with H5 and GH5 .

• Multiple antibody comparison: Using different antibodies targeting distinct epitopes of H1.0 can confirm specificity through concordant results.

• Quantitative binding assays: Affinity binding assays showing a KD of 3.3 x 10-9 for H1F0 peptide have been used to validate commercial antibodies . Similar approaches can be employed to assess binding kinetics in experimental systems.

• Western blot verification: Confirming a single band of the expected molecular weight in Western blot analysis of cell lysates (e.g., NIH3T3, A549) provides further validation of specificity .

What methods can researchers use to study tissue-specific variations in Histone H1.0 expression?

Several methodological approaches can be employed to investigate tissue-specific variations in Histone H1.0 expression:

• Single-cell RNA sequencing (scRNA-seq): This technique has revealed that H1.0 is more highly expressed than other linker histone variants in fibroblasts from various organs. scRNA-seq datasets from human hearts have shown positive correlations between histone H1.0 and periostin, a canonical marker of fibroblast activation .

• Genotype-Tissue Expression (GTEx) project data analysis: Researchers have used GTEx data to examine correlations between H1.0 expression and tissue-specific markers, revealing tissue-specific expression patterns .

• Immunohistochemistry on tissue microarrays: Using validated antibodies with optimized protocols on tissue microarrays allows for comparison of H1.0 expression across multiple tissue types simultaneously. High-pressure and temperature sodium citrate buffer (pH 6.0) has been validated for antigen retrieval .

• Fluorescence microscopy with quantitative image analysis: This approach allows visualization of H1.0 distribution within tissues and cells, with quantification of expression levels through fluorescence intensity measurements.

• Chromatin immunoprecipitation followed by sequencing (ChIP-seq): This method can map the genomic distribution of H1.0 across different tissue types, providing insight into tissue-specific roles in gene regulation.

• Global transcriptome analyses: Studies in genetically diverse populations, such as mice administered isoproterenol, have shown associations between H1.0 levels and tissue-specific pathologies (e.g., heart muscle dysfunction) .

• Protein extraction and Western blotting: Using optimized extraction protocols for different tissue types followed by Western blotting with a 1:1,000 dilution of validated H1.0 antibodies provides quantitative comparison of protein levels .

What are common sources of non-specific binding when using Histone H1.0 antibodies, and how can they be mitigated?

Non-specific binding is a common challenge when working with Histone H1.0 antibodies. Researchers can identify and address several potential sources:

• Cross-reactivity with other histone variants: Some antibodies targeting residues 27-30 in H1.0's globular domain cross-react with H5 and GH5 . To mitigate:

  • Select antibodies specifically validated against cross-reactivity (e.g., those targeting residues 24-27)

  • Include peptide blocking controls to verify specificity

  • Consider using recombinant monoclonal antibodies rather than polyclonal antibodies

• Conformational epitope recognition: Some antibodies recognize three-dimensional conformational antigenic determinants rather than linear sequences . To address:

  • Ensure proper protein folding is maintained during sample preparation

  • Use multiple fixation protocols to determine optimal epitope preservation

  • Consider native versus denaturing conditions when appropriate

• Fc receptor binding in tissues: To reduce:

  • Include Fc receptor blocking reagents in immunohistochemistry protocols

  • Use Fab or F(ab')2 fragments instead of whole IgG when necessary

  • Implement appropriate blocking with BSA (0.05%) as validated in commercial protocols

• Improper antigen retrieval: For paraffin-embedded samples:

  • Use validated high-pressure and temperature sodium citrate buffer (pH 6.0)

  • Optimize retrieval time and temperature for specific tissue types

  • Consider alternative retrieval methods if background remains high

• Secondary antibody cross-reactivity: To minimize:

  • Use secondary antibodies pre-adsorbed against tissue species

  • Include secondary-only controls

  • Optimize secondary antibody concentration

How can researchers quantitatively assess Histone H1.0 levels across different experimental conditions?

Quantitative assessment of Histone H1.0 requires robust methodology suited to the experimental question. Several approaches have been validated:

• Western blotting with densitometry:

  • Use validated 1:1,000 dilution of primary antibody

  • Include appropriate loading controls (e.g., total histone H3)

  • Employ linear range detection methods

  • Normalize to total protein using stain-free technology or housekeeping proteins

  • Analyze using software like ImageJ with appropriate background subtraction

• Quantitative immunofluorescence microscopy:

  • Use consistent image acquisition parameters

  • Include calibration standards

  • Employ nuclear counterstains to normalize signal

  • Use automated image analysis software to measure nuclear signal intensity

  • Calculate nucleus-to-cytoplasm ratios to control for background

• Chromatin immunoprecipitation followed by qPCR (ChIP-qPCR):

  • Implement spike-in controls for normalization

  • Use percent input method for quantification

  • Include IgG controls

  • Validate primers for specific genomic regions of interest

  • Apply appropriate statistical analysis for comparing conditions

• Mass spectrometry-based proteomics:

  • Use stable isotope labeling (SILAC, TMT, iTRAQ)

  • Implement targeted approaches (PRM, MRM) for greater sensitivity

  • Include internal standard peptides

  • Account for extraction efficiency differences between samples

  • Use specialized software for histone PTM quantification

• Flow cytometry:

  • Optimize cell permeabilization for nuclear antigen access

  • Include fluorescence minus one (FMO) controls

  • Use median fluorescence intensity (MFI) for quantification

  • Apply compensation when using multiple fluorophores

  • Gate on specific cell populations of interest

These methodologies provide complementary information about H1.0 levels and should be selected based on the specific experimental question and available resources.