Mono-methyl-H1F0 (K101) Antibody

What is Mono-methyl-H1F0 (K101) Antibody and what specific modification does it detect?

Mono-methyl-H1F0 (K101) Antibody is a polyclonal antibody raised in rabbits that specifically recognizes the mono-methylation modification at lysine 101 (K101) of the human Histone H1.0 protein (H1F0). This antibody targets a post-translational modification that may play significant roles in chromatin structure regulation and cellular differentiation . The specificity for this single methylation site allows researchers to investigate site-specific epigenetic modifications of linker histones, as opposed to core histones which have been more extensively studied.

What are the technical specifications and validated applications of this antibody?

The Mono-methyl-H1F0 (K101) Antibody is available as an unconjugated primary polyclonal antibody produced in rabbits with IgG isotype . It has been experimentally validated for multiple applications:

The antibody has been specifically tested with reactivity to human (Homo sapiens) samples, with validation experiments conducted in cell lines including HepG2 and HeLa .

What is the biological significance of Histone H1.0 in cellular function?

Histone H1.0 (H1F0) is a linker histone necessary for the condensation of nucleosome chains into higher-order chromatin structures. Unlike other H1 variants, H1F0 is predominantly found in cells that are in terminal stages of differentiation or that exhibit low rates of cell division . Recent research has revealed that H1F0 plays a critical role in coupling cellular mechanical behaviors to chromatin structure and regulating cellular stress responses .

H1F0 has been shown to be necessary for:

• TGF-β-induced contractile phenotypes in fibroblasts

• Fibroblast proliferation in response to growth factors

• Cell migration in wound healing assays

• Mechano-transduction between extracellular stress and nuclear responses

How should I design ChIP experiments using this antibody for optimal results?

When designing ChIP experiments with Mono-methyl-H1F0 (K101) Antibody, consider the following methodology based on validated protocols:

• Sample preparation: Treat cells (4×10^6 recommended) with Micrococcal Nuclease to fragment chromatin while preserving protein-DNA interactions, followed by sonication to create appropriately sized DNA fragments (200-500bp) .

• Immunoprecipitation: Use 5μg of anti-H1F0 (K101me1) antibody per ChIP reaction, alongside a control normal rabbit IgG sample processed in parallel .

• DNA recovery and analysis: Following IP, purify DNA and quantify enrichment using real-time PCR. Validated protocols have successfully used primers against the β-Globin promoter for quantification .

• Controls: Always include:

  • Input chromatin (pre-immunoprecipitation sample)

  • IgG control (normal rabbit IgG)

  • Positive control regions (where H1F0 binding is expected)

  • Negative control regions (where H1F0 binding is not expected)

What sample preparation protocols maximize signal strength in immunofluorescence applications?

For optimal immunofluorescence results with this antibody, follow this validated protocol:

• Fixation: Fix cells in 4% formaldehyde for 15 minutes at room temperature .

• Permeabilization: Permeabilize using 0.2% Triton X-100 for 10 minutes to allow antibody access to nuclear targets .

• Blocking: Block in 10% normal goat serum for 30 minutes at room temperature to reduce non-specific binding .

• Primary antibody incubation: Incubate with Mono-methyl-H1F0 (K101) antibody at 1:2.5-1:10 dilution overnight at 4°C .

• Secondary antibody: Use Alexa Fluor 488-conjugated AffiniPure Goat Anti-Rabbit IgG(H+L) for detection. Include DAPI counterstaining for nuclear visualization .

This protocol has been successfully used with HepG2 cells and provides clear nuclear localization patterns consistent with the expected distribution of histone H1.0 .

How can this antibody be integrated into studies investigating the relationship between mechanical stress and chromatin structure?

Based on recent findings about H1F0's role in mechanical behaviors , researchers can:

• Mechanical stress experiments: Design experiments applying mechanical stress (stretch, compression, or shear) to fibroblasts or other relevant cells, followed by immunostaining with Mono-methyl-H1F0 (K101) antibody to examine changes in methylation patterns.

• Traction force correlation: Use the antibody in combination with traction force microscopy to correlate H1F0 methylation status with cellular force generation. This approach can build upon validated methods where cells are seeded onto fluorescently labeled BSA beads to measure deformation .

• Contraction assays: Implement gel contraction assays following the methodology described in recent literature , comparing control and treatment conditions while monitoring H1F0 K101 methylation status using the antibody in parallel samples.

• Integrated analysis with gene expression: Combine immunofluorescence or ChIP using this antibody with RNA-seq or qPCR to correlate H1F0 methylation with expression of genes involved in extracellular matrix, cytoskeletal dynamics, and contractile functions.

How does mono-methylation of H1F0 at K101 potentially affect its role in genome organization?

While direct research on K101 methylation effects is still emerging, based on the known functions of H1F0 and histone methylation principles, researchers can investigate:

• Chromatin compaction effects: Mono-methylation at K101 likely modulates H1F0's ability to condense chromatin, potentially altering the higher-order structure of differentiated cell chromatin. This could be analyzed through combination of ChIP-seq using this antibody with techniques like ATAC-seq to assess chromatin accessibility.

• Interaction partners: The methylation may create or disrupt binding interfaces for chromatin-modifying complexes. Researchers could employ this antibody in combination with proximity ligation assays or co-immunoprecipitation to identify proteins that specifically recognize the K101 methylated form of H1F0.

• Genomic localization: Research suggests H1F0 depletion affects H3K27 acetylation at specific loci . Investigators can use this antibody in ChIP-seq experiments to map genomic distribution of K101-methylated H1F0 and correlate this with histone modification patterns and gene expression changes.

• Cell differentiation dynamics: Given H1F0's enrichment in terminally differentiated cells, the K101 methylation may serve as a marker or functional component of differentiation programs, which could be tracked during cellular differentiation processes.

What strategies can be employed to study the relationship between H1F0 K101 methylation and cellular mechanical behaviors?

Building on recent findings that H1F0 couples cellular mechanical behaviors to chromatin structure , researchers can implement:

• Methylation-specific knockin models: Generate cell lines expressing mutant H1F0 where K101 is replaced with amino acids that either prevent methylation (K101R) or mimic constitutive methylation (K101M), then assess mechanical behaviors using traction force microscopy and gel contraction assays.

• Methyltransferase identification: Use this antibody in cellular assays combined with knockdown of candidate methyltransferases to identify the enzyme responsible for K101 methylation, potentially revealing a mechanosensitive regulatory pathway.

• Mechanical stimulation time course: Apply defined mechanical forces to cells and use the antibody to track temporal changes in K101 methylation, potentially identifying this modification as an early or late response to mechanical cues.

• Cross-tissue analysis: Compare K101 methylation patterns in fibroblasts from different tissues (cardiac, lung, skin) that exhibit varying mechanical properties, as these have been shown to depend on H1F0 for their activation .

How might this antibody be used to study the role of H1F0 in fibrosis and tissue remodeling?

Recent research indicates H1F0 depletion prevents fibrosis in cardiac muscle . Researchers can leverage this antibody to:

• Fibrosis model analysis: In animal models of fibrosis (cardiac, pulmonary, hepatic, etc.), use this antibody for immunohistochemistry to correlate K101 methylation status with progression of fibrotic changes.

• TGF-β signaling intersection: Investigate how TGF-β treatment, a canonical activator of fibroblasts, affects K101 methylation dynamics using Western blot and immunofluorescence with this antibody.

• Therapeutic target validation: In intervention studies using anti-fibrotic agents, monitor changes in H1F0 K101 methylation as a potential biomarker of treatment efficacy.

• Cell-specific effects: Combine this antibody with markers for specific cell types in fibrotic tissues to determine which cell populations exhibit changes in H1F0 K101 methylation during disease progression.

What are common technical issues when using this antibody and how can they be addressed?

Researchers may encounter several challenges when working with Mono-methyl-H1F0 (K101) Antibody:

• High background in immunofluorescence:

  • Issue: Diffuse cytoplasmic staining rather than specific nuclear signal

  • Solution: Optimize fixation conditions (reduce time), increase blocking duration to 1 hour, and carefully titrate antibody concentration. Try 1:10 dilution before moving to more concentrated preparations .

• Weak signal in Western blot:

  • Issue: Faint bands at expected 21 kDa size

  • Solution: Load more protein (20-30 μg), reduce washing stringency, increase antibody concentration (1:50 dilution recommended for challenging samples), and extend primary antibody incubation to overnight at 4°C .

• Multiple bands in Western blot:

  • Issue: Additional bands beyond expected 21 kDa

  • Solution: These may represent degradation products or cross-reactivity. Validate specificity using peptide competition assays with the immunizing peptide containing mono-methylated K101.

• ChIP efficiency issues:

  • Issue: Low enrichment over IgG background

  • Solution: Optimize chromatin fragmentation, increase antibody amount to 7-10 μg per reaction, extend incubation time, and ensure proper handling of chromatin to preserve methylation status.

How can the specificity of this antibody for mono-methylated versus unmethylated or other methylated forms be validated?

To confirm the specificity for mono-methylated K101:

• Peptide competition assays: Perform parallel experiments with the antibody pre-incubated with:

  • Mono-methylated K101 peptide (should abolish signal)

  • Unmethylated K101 peptide (should not affect signal)

  • Di- or tri-methylated K101 peptides (should not affect signal)

• Methyltransferase manipulation: Knockdown/knockout or overexpress methyltransferases that target H1F0, then assess antibody signal changes by Western blot and immunofluorescence.

• Mass spectrometry validation: Perform immunoprecipitation with the antibody followed by mass spectrometry to confirm that the captured protein contains mono-methylation at K101.

• Demethylase treatment: Treat nuclear extracts with histone demethylases prior to Western blot analysis to assess signal reduction.

How should researchers interpret seemingly contradictory results between applications (e.g., positive WB but negative IF)?

When facing application-specific discrepancies:

• Epitope accessibility differences: The K101 methylation site may be masked in certain applications. For negative IF results despite positive WB, try alternative fixation methods (methanol instead of paraformaldehyde) or gentler permeabilization to preserve epitope accessibility.

• Protein concentration thresholds: Western blot can concentrate proteins, making detection easier than in situ techniques. If WB is positive but IF negative, try increasing antibody concentration for IF (1:1 dilution) and extend incubation times.

• Cell type-specific methylation: Different cell types may have varying levels of K101 methylation. Compare results across multiple cell lines, particularly using HepG2 cells where this antibody has been validated .

• Modification stability: The methylation may be lost during sample processing for certain applications. Include freshly prepared samples alongside stored ones to assess potential degradation of the modification.

How does this antibody complement studies of other histone modifications and chromatin dynamics?

This antibody can be integrated with broader epigenetic investigations through:

• Sequential ChIP (Re-ChIP): Perform ChIP first with Mono-methyl-H1F0 (K101) antibody followed by a second IP with antibodies against other modifications (e.g., H3K27ac, which has been linked to H1F0 function ) to identify genomic regions with co-occurrence of modifications.

• Multiplexed immunofluorescence: Combine this antibody with antibodies against core histone modifications or chromatin remodeling factors to visualize spatial relationships in the nucleus.

• Correlation with nucleosome positioning: Integrate ChIP-seq data from this antibody with MNase-seq to determine how K101 methylation affects nucleosome organization and spacing.

• Integration with chromosome conformation capture: Combine this antibody's ChIP with Hi-C or related techniques to investigate how K101 methylation influences 3D genome organization, particularly in differentiated cells where H1F0 is abundant.

How can this antibody contribute to investigating interactions between mechanical stimuli and epigenetic regulation?

Building on findings that H1F0 mediates mechanical responses in cells , researchers can:

• Mechanically stressed chromatin immunoprecipitation: Apply defined mechanical forces to cells (stretch, compression, fluid shear) followed by ChIP with this antibody to map changes in genomic distribution of K101-methylated H1F0.

• Live-cell imaging: Develop fluorescently-tagged nanobodies based on this antibody's binding characteristics to visualize real-time changes in H1F0 K101 methylation during application of mechanical forces.

• Micropatterned substrates: Seed cells on substrates with varying stiffness or geometric constraints, then use this antibody to correlate mechanical inputs with K101 methylation patterns.

• Multi-omics integration: Combine ChIP-seq using this antibody with transcriptomics and proteomics from cells under varying mechanical conditions to construct comprehensive regulatory networks linking mechanical stimuli to epigenetic changes and downstream effects.

What experimental strategies can integrate this antibody with therapeutic development for fibrotic disorders?

Given H1F0's role in fibrosis , researchers can utilize this antibody to:

• Drug screening assays: Develop high-content screening assays using this antibody to identify compounds that alter H1F0 K101 methylation status in activated fibroblasts.

• Pharmacodynamic biomarker development: In animal models treated with anti-fibrotic agents (similar to monomethyl fumarate mentioned in the literature ), use this antibody to assess changes in H1F0 K101 methylation as a potential response biomarker.

• Target validation: Use CRISPR-based approaches to modify the K101 residue to non-methylatable forms, then assess fibrotic responses. This antibody would serve as a control to confirm loss of the modification.

• Patient-derived samples: Apply this antibody in immunohistochemistry of fibrotic tissue samples from patients to assess correlation between K101 methylation levels and disease severity or treatment response.