Mono-methyl-Histone H3.1 (R17) Recombinant Monoclonal Antibody

What is Mono-methyl-Histone H3.1 (R17) and why is it significant in epigenetic research?

Mono-methyl-Histone H3.1 (R17) refers to histone H3.1 protein that is mono-methylated at the arginine 17 position. This post-translational modification is involved in transcriptional regulation, chromatin structure modulation, DNA repair processes, cellular identity determination, and epigenetic signaling pathways . The significance of this modification lies in its role in controlling gene expression through altering chromatin accessibility. The recombinant monoclonal antibody targeting this specific modification allows researchers to study these processes with high specificity and sensitivity, making it an essential tool for epigenetic research. When designing experiments, researchers should consider that this modification exists in a complex network of histone modifications that collectively regulate gene expression.

What applications has the Mono-methyl-Histone H3.1 (R17) Recombinant Monoclonal Antibody been validated for?

The Mono-methyl-Histone H3.1 (R17) Recombinant Monoclonal Antibody has been validated for multiple applications in molecular and cellular biology research. According to available data, this antibody has been successfully used in ELISA, Western Blot (WB), Immunocytochemistry (ICC), and Immunofluorescence (IF) applications . For Western Blot, the recommended dilution ranges from 1:500 to 1:2000; for Immunofluorescence, the recommended dilution is 1:50 to 1:500; and for Immunocytochemistry, the recommended dilution is also 1:50 to 1:500 . These dilution ranges should be optimized by researchers depending on their specific experimental conditions, sample types, and detection methods employed. The versatility of this antibody across multiple applications makes it valuable for researchers investigating histone modifications from different methodological approaches.

What is the species reactivity of this antibody and how does this influence experimental design?

The Mono-methyl-Histone H3.1 (R17) Recombinant Monoclonal Antibody has been specifically designed to react with human (Homo sapiens) proteins . This specificity means that researchers working with human cell lines, tissues, or clinical samples will achieve optimal results. When designing experiments, researchers should be aware that this antibody might not recognize the equivalent modifications in other species or might show reduced affinity, which could lead to weaker signals or false negatives in non-human samples. For cross-species studies, validation experiments should be performed to confirm reactivity, or species-specific antibodies should be considered as alternatives. The human-specific nature of this antibody makes it particularly valuable for studies focusing on human diseases and development where epigenetic modifications play important roles.

What are the recommended storage conditions for maintaining antibody activity?

To maintain optimal activity of the Mono-methyl-Histone H3.1 (R17) Recombinant Monoclonal Antibody, proper storage conditions are essential. The antibody should be aliquoted upon receipt to avoid repeated freeze-thaw cycles and stored at -20°C . The antibody is typically supplied in PBS (pH 7.4) containing 150 mM NaCl, 0.02% sodium azide, and 50% glycerol as a stabilizer . When working with the antibody, it should be thawed on ice or at 4°C rather than at room temperature to preserve its binding capacity. Repeated freeze-thaw cycles can lead to protein denaturation and loss of antibody specificity and sensitivity. For long-term storage of working dilutions, addition of carrier proteins such as BSA (0.1-1%) may help maintain stability. Antibody activity should be monitored periodically, especially after extended storage periods, to ensure consistent experimental results.

How does H3.1 function as a chromatin redox sensor and what implications does this have for interpreting results?

Recent research has revealed that histone H3.1 functions as a chromatin-embedded redox sensor due to its unique cysteine residue at position 96 (Cys96), which is absent in other H3 variants . This residue can be oxidized in response to hydrogen peroxide (H₂O₂), particularly nuclear H₂O₂ (nH₂O₂), leading to nucleosome instability and subsequent exchange of H3.1 for the H3.3 variant . Studies have demonstrated that nH₂O₂-dependent oxidation of H3.1 Cys96 promotes this histone exchange, which is associated with chromatin decompaction, increased accessibility at promoter regions, and activation of gene expression, particularly genes involved in epithelial-mesenchymal transition (EMT) .

When interpreting results involving H3.1 modifications, researchers should consider that oxidative stress conditions might alter the chromatin landscape by promoting H3.1 exchange, potentially influencing other histone modifications including R17 methylation. Experimental designs should account for redox states in the cellular environment, and controls for oxidative conditions should be included. The dynamic interplay between redox signaling and histone modifications represents an important layer of epigenetic regulation that may affect experimental outcomes and interpretation of results involving H3.1 modifications.

What methodological approaches can be used to distinguish between different methylation states of H3.1 R17?

Distinguishing between different methylation states of H3.1 R17 (unmethylated, mono-methylated, di-methylated, and tri-methylated) requires careful methodological approaches. The Mono-methyl-Histone H3.1 (R17) Recombinant Monoclonal Antibody is specifically designed to recognize only the mono-methylated form at R17 . To ensure specificity, researchers should:

• Perform peptide competition assays using unmethylated, mono-methylated, di-methylated, and tri-methylated peptides containing the R17 residue.

• Include positive controls (cells or tissues known to have high levels of H3.1R17me1) and negative controls (cells with CARM1/PRMT4 knockdown, as these are the primary methyltransferases for this site).

• Use mass spectrometry-based approaches for unbiased detection of different methylation states.

• Consider employing parallel antibodies specific to other methylation states for comparative analysis.

• Validate results using orthogonal methods such as ChIP-seq followed by mass spectrometry.

These methodological approaches help ensure that the signals detected truly represent mono-methylation at R17 rather than other methylation states or modifications at nearby residues, increasing the reliability of experimental findings.

How can ChIP-seq experiments be optimized when using the Mono-methyl-Histone H3.1 (R17) antibody?

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a powerful technique for mapping histone modifications genome-wide. When using the Mono-methyl-Histone H3.1 (R17) antibody for ChIP-seq, several optimization strategies should be considered:

• Cross-linking optimization: Standard 1% formaldehyde for 10 minutes at room temperature works for most histone modifications, but optimal cross-linking conditions may vary for R17 methylation.

• Sonication parameters: Optimize sonication conditions to yield DNA fragments between 200-600 bp, while avoiding excessive heat that could affect epitope recognition.

• Antibody validation: Confirm antibody specificity using peptide competition assays specifically for ChIP applications.

• Input controls: Always include input DNA controls and IgG negative controls.

• Sequential ChIP: Consider sequential ChIP (ChIP-reChIP) to identify genomic regions containing both H3.1R17me1 and other histone marks of interest.

Based on research findings in the field, ChIP-seq experiments with H3.1 variant-specific antibodies have successfully identified differential occupancy patterns at transcription start sites, with H3.1 showing decreased occupancy and H3.3 showing increased occupancy after hydrogen peroxide treatment . This indicates that the H3.1 methylation patterns may be dynamically regulated in response to cellular stressors, which should be considered when designing ChIP-seq experiments with the Mono-methyl-Histone H3.1 (R17) antibody.

What is the relationship between H3.1 R17 methylation and other histone modifications in the histone code?

H3.1 R17 mono-methylation exists within a complex network of histone modifications collectively known as the histone code. The relationship between H3.1 R17 methylation and other modifications is multifaceted:

• Cross-talk with nearby modifications: Methylation at R17 may influence or be influenced by other modifications on the H3 tail, particularly those at K9, K14, and R2. For example, acetylation at K14 may facilitate enzyme access to R17 for methylation.

• Exclusivity with certain modifications: Some modifications may be mutually exclusive with R17 methylation due to steric hindrance or enzyme competition.

• Sequential modification patterns: R17 methylation may be part of sequential modification patterns, where one modification leads to another in a specific order.

• Functional consequences: The combination of R17 methylation with other modifications determines functional outcomes such as transcriptional activation or repression.

When investigating H3.1 R17 methylation, researchers should consider analyzing co-occurring modifications to understand the broader epigenetic context. Methods such as sequential ChIP or mass spectrometry-based approaches can help identify modification patterns that co-exist with R17 methylation. Understanding these relationships is crucial for interpreting the biological significance of R17 methylation in different cellular contexts and disease states.

What are common issues encountered in Western blot applications with this antibody and how can they be resolved?

When using the Mono-methyl-Histone H3.1 (R17) Recombinant Monoclonal Antibody in Western blot applications, researchers may encounter several challenges. Here are common issues and their solutions:

When optimizing Western blots with this antibody, researchers should be particularly attentive to extraction methods that preserve histone modifications, as harsh conditions may affect the methylation status. Additionally, including appropriate positive controls (such as recombinant H3.1 with verified R17 mono-methylation) and negative controls (such as samples treated with demethylase enzymes) can help validate the specificity of the observed signals.

How can specificity of the antibody be verified in different experimental contexts?

Verifying the specificity of the Mono-methyl-Histone H3.1 (R17) Recombinant Monoclonal Antibody across different experimental contexts is crucial for reliable results. Here are methodological approaches for verification:

• Peptide competition assays: Pre-incubate the antibody with excess mono-methylated H3.1 R17 peptide before application. A specific antibody will show significantly reduced signal when pre-bound to its target epitope.

• Methyltransferase inhibition or knockout: Treat cells with inhibitors of arginine methyltransferases (particularly CARM1/PRMT4) or use cells with knocked-out methyltransferases. Reduced signal confirms specificity for the methylated form.

• Demethylase overexpression: Overexpress histone arginine demethylases that target R17. Decreased signal supports antibody specificity.

• Mass spectrometry validation: Immunoprecipitate with the antibody and analyze by mass spectrometry to confirm that the captured proteins contain the expected modification.

• Cross-reactivity testing: Test the antibody against a panel of similar histone modifications, especially di- and tri-methylated R17, as well as methylations at other arginine residues (R2, R8, R26) to ensure specificity.

• Dot blot analysis: Perform dot blots with modified and unmodified peptides to establish binding specificity without the complications of protein denaturation or complex sample matrices.

These validation approaches should be applied within the specific experimental context (cell type, treatment conditions, etc.) being studied, as antibody performance can vary across different biological systems and experimental conditions.

What considerations should be made when using this antibody in immunofluorescence applications?

When using the Mono-methyl-Histone H3.1 (R17) Recombinant Monoclonal Antibody for immunofluorescence applications, several important considerations should be addressed:

• Fixation method: Different fixation methods can affect epitope accessibility. For histone modifications, 4% paraformaldehyde (10-15 minutes) is generally recommended, but methanol fixation may provide better nuclear penetration. Compare both methods to determine optimal conditions.

• Permeabilization: Adequate permeabilization is crucial for nuclear antigens. Try 0.1-0.5% Triton X-100 for 10 minutes, but optimize based on cell type.

• Antibody dilution: Begin with the recommended dilution range (1:50-1:500) and optimize. Too high concentration may lead to non-specific binding, while too low may result in weak signals.

• Blocking conditions: Use 5% normal serum from the same species as the secondary antibody. BSA (3-5%) can also be effective. Block for at least 1 hour at room temperature.

• Controls: Include a negative control (no primary antibody) and positive controls (cells known to express H3.1R17me1). Consider using peptide competition controls to demonstrate specificity.

• Signal detection optimization: Adjust exposure times to capture specific signals while avoiding saturation. Use appropriate filter sets matched to your secondary antibody fluorophores.

• Co-localization studies: When performing co-localization with other nuclear markers, select secondary antibodies with well-separated emission spectra to avoid bleed-through.

• Quantification approaches: For quantitative analyses, establish consistent imaging parameters and use appropriate software for signal quantification.

By addressing these considerations methodically, researchers can achieve reliable and reproducible immunofluorescence results with the Mono-methyl-Histone H3.1 (R17) antibody.

How does the exchange between H3.1 and H3.3 variants influence gene expression and cellular phenotypes?

The exchange between histone variants H3.1 and H3.3 represents a dynamic mechanism for regulating chromatin structure and gene expression. Research has revealed several key aspects of this process:

• Differential incorporation mechanisms: H3.1 incorporation is primarily replication-dependent, whereas H3.3 incorporation is replication-independent . This difference allows for dynamic regulation of chromatin structure throughout the cell cycle.

• Chromatin decompaction: The replacement of H3.1 with H3.3 is associated with chromatin decompaction and increased accessibility at promoter regions . Electron microscopy studies have shown that this exchange reduces heterochromatin content, particularly near the nuclear envelope .

• Transcriptional activation: H3.3 incorporation correlates with transcriptional activation . The exchange from H3.1 to H3.3 at promoter regions facilitates the binding of transcription factors and the assembly of the transcriptional machinery.

• Redox sensitivity: The unique cysteine residue (Cys96) in H3.1 serves as a redox sensor, with oxidation promoting H3.1 exchange for H3.3 . This mechanism links oxidative signaling to epigenetic reprogramming.

• EMT regulation: Research has shown that the exchange of H3.1 for H3.3 precedes and is required for epithelial-mesenchymal transition (EMT), a process crucial for cancer metastasis . This exchange activates EMT gene expression programs driven by nuclear hydrogen peroxide.

• Temporal dynamics: The exchange shows specific temporal dynamics, with peak H3.3 incorporation at promoters occurring around 4 hours after stimulus, followed by a return toward baseline levels by 24 hours . This indicates a transitional role in gene activation rather than a permanent change.

Understanding the interplay between H3.1 R17 methylation and these variant exchange dynamics presents an important area for future research, as both processes contribute to epigenetic regulation of gene expression and cellular phenotypes.

What role does H3.1 R17 mono-methylation play in disease processes and potential therapeutic targets?

H3.1 R17 mono-methylation plays significant roles in various disease processes, particularly through its influence on gene expression regulation:

• Cancer progression: Aberrant histone arginine methylation, including at R17, has been implicated in cancer development and progression. Changes in methylation patterns can lead to inappropriate activation or silencing of genes involved in cell proliferation, apoptosis, and metastasis.

• Inflammatory diseases: Dysregulation of histone arginine methylation contributes to aberrant inflammatory responses by affecting the expression of cytokines and other immune-related genes.

• Neurodegenerative disorders: Altered histone methylation patterns have been observed in various neurodegenerative conditions, potentially affecting neuronal gene expression programs.

• Developmental disorders: As histone modifications are crucial for proper gene expression during development, abnormal R17 methylation may contribute to developmental abnormalities.

From a therapeutic perspective, several approaches targeting histone arginine methylation are being explored:

• Methyltransferase inhibitors: Compounds targeting the enzymes responsible for R17 methylation (primarily CARM1/PRMT4) could normalize aberrant methylation patterns.

• Demethylase activators: Molecules that enhance the activity of arginine demethylases might counter excessive methylation at R17.

• Reader domain inhibitors: Blocking the proteins that recognize and bind to methylated R17 could interrupt downstream signaling without altering the methylation itself.

• Combination epigenetic therapies: Targeting multiple epigenetic modifications simultaneously may provide synergistic effects in diseases with complex epigenetic dysregulation.

Researchers using the Mono-methyl-Histone H3.1 (R17) antibody can contribute to this field by characterizing methylation patterns in disease models, identifying genes regulated by this modification, and evaluating the effects of potential therapeutic compounds on R17 methylation levels and distribution.

How do the functions of H3.1 R17 mono-methylation compare with other arginine methylation sites on histones?

H3.1 R17 mono-methylation is one of several arginine methylation sites on histones, each with distinct and sometimes overlapping functions. Understanding these comparisons provides important context for research:

Key functional comparisons include:

• Cooperation and antagonism: H3R17 methylation often cooperates with H3R26 methylation (both mediated by CARM1/PRMT4) but may antagonize H3R2 methylation (mediated by PRMT6) in some contexts.

• Cross-talk with other modifications: Unlike H3R2 methylation, which can inhibit H3K4 methylation, H3R17 methylation often positively correlates with nearby activating modifications like H3K4 methylation and H3K9 acetylation.

• Temporal dynamics: H3R17 methylation often occurs rapidly in response to signaling events (particularly hormone signaling), while other arginine methylations may show different temporal patterns.

• Reader proteins: Different methylarginine sites are recognized by distinct reader proteins, leading to recruitment of different downstream effector complexes.

Researchers studying H3.1 R17 mono-methylation should consider these comparative aspects when designing experiments and interpreting results, as the specific biological context may influence the functional outcomes of this modification.

What emerging technologies can enhance the detection and functional analysis of H3.1 R17 mono-methylation?

Several cutting-edge technologies are expanding our capabilities for studying H3.1 R17 mono-methylation:

• CUT&RUN and CUT&Tag: These techniques offer advantages over traditional ChIP-seq by providing higher signal-to-noise ratios and requiring fewer cells. They can be optimized for the Mono-methyl-Histone H3.1 (R17) antibody to map genomic distributions with greater sensitivity.

• Single-cell epigenomics: Techniques such as single-cell CUT&Tag allow for analysis of histone modification heterogeneity at the single-cell level, revealing cell-to-cell variation in H3.1 R17 methylation patterns within populations.

• Proximity ligation assays (PLA): These can detect interactions between H3.1 R17 methylation and other chromatin-associated proteins in situ, providing spatial context within the nucleus.

• Live-cell imaging of histone modifications: Using techniques such as FRAP (Fluorescence Recovery After Photobleaching) with specific readers of H3.1 R17 methylation fused to fluorescent proteins can reveal dynamics of this modification in living cells.

• Mass spectrometry-based approaches: Advanced mass spectrometry methods such as MALDI-TOF MS/MS and quantitative proteomics can identify and quantify combinatorial patterns of histone modifications co-occurring with H3.1 R17 methylation.

• CRISPR-based epigenome editing: Tools like dCas9 fused to CARM1/PRMT4 can induce site-specific H3.1 R17 methylation, allowing for causal studies of this modification's effects on gene expression and cellular phenotypes.

• Computational integration approaches: Machine learning algorithms can integrate multiple epigenomic datasets to predict functional consequences of H3.1 R17 methylation patterns across the genome.

These emerging technologies offer researchers powerful new approaches to understanding the dynamics, distribution, and functional significance of H3.1 R17 mono-methylation in various biological contexts.

What are the key experimental controls required for studying H3.1 R17 mono-methylation in different research contexts?

Robust experimental controls are essential for reliable research on H3.1 R17 mono-methylation. Here are key controls that should be implemented across different research contexts:

• Antibody specificity controls:

  • Peptide competition assays using mono-methylated H3R17 peptides versus unmethylated and differently methylated peptides

  • Dot blots or Western blots with recombinant histones containing defined modifications

  • Use of alternative antibodies targeting the same modification for validation

• Biological manipulation controls:

  • CARM1/PRMT4 knockdown or knockout (enzymes responsible for H3R17 methylation)

  • CARM1/PRMT4 overexpression to increase modification levels

  • Treatment with methyltransferase inhibitors

  • Demethylase overexpression

• ChIP-seq specific controls:

  • Input DNA controls to account for genomic biases

  • IgG controls to establish background binding

  • Total H3 ChIP to normalize for nucleosome occupancy

  • Sequential ChIP to verify co-occurrence with other marks

• Immunofluorescence controls:

  • Secondary antibody-only controls

  • Peptide competition controls

  • Positive controls (cells known to have the modification)

  • Negative controls (cells with CARM1/PRMT4 knockdown)

• Functional analysis controls:

  • Point mutations in H3.1 at R17 (R17K or R17A) to prevent methylation

  • Time-course experiments to establish temporal dynamics

  • Dose-response studies for treatments affecting methylation levels

• Cross-species validation:

  • Parallel experiments in different cell lines/organisms to establish conservation

  • Controls for histone variant-specific effects

Implementation of these controls helps distinguish specific effects of H3.1 R17 mono-methylation from other biological processes and technical artifacts, enhancing the reliability and interpretability of research findings.

How can the study of H3.1 R17 mono-methylation contribute to our understanding of epigenetic inheritance and cellular memory?

The study of H3.1 R17 mono-methylation offers unique insights into epigenetic inheritance and cellular memory due to several key properties:

• Replication dynamics: As H3.1 is primarily incorporated during DNA replication, understanding how R17 methylation patterns are maintained or altered during cell division can illuminate mechanisms of epigenetic inheritance. Research questions include:

  • How are methylation patterns distributed to daughter cells during replication?

  • What role do chaperone proteins play in preserving R17 methylation during nucleosome assembly?

• Stability and turnover: The stability of H3.1 R17 mono-methylation over time influences its potential as a carrier of epigenetic information. Studies can examine:

  • The half-life of this modification compared to other histone marks

  • The enzymes responsible for active removal versus passive dilution during replication

  • How cellular stressors affect the stability of this modification

• Developmental programming: H3.1 R17 methylation patterns established during development may contribute to cell fate decisions and cellular memory. Researchers can investigate:

  • Changes in methylation patterns during differentiation

  • The role of R17 methylation in maintaining cell identity

  • The potential for these marks to be disrupted in disease states

• Environmental responses: How H3.1 R17 methylation responds to environmental signals can inform our understanding of cellular adaptation and memory. Key areas include:

  • The dynamics of R17 methylation in response to signaling pathways

  • The persistence of environmentally-induced changes in methylation

  • The role of this modification in adaptive responses to repeated stimuli

• Cross-generational inheritance: While most histone modifications are reprogrammed during gametogenesis, some may persist. Research could explore:

  • Whether R17 methylation patterns can escape reprogramming

  • The potential role of this modification in transgenerational epigenetic inheritance

  • Interactions with other epigenetic mechanisms like DNA methylation

By addressing these research areas, scientists can better understand how H3.1 R17 mono-methylation contributes to the complex landscape of epigenetic inheritance and cellular memory, with implications for development, aging, and disease.