Mono-methyl-HIST1H1E (K63) Antibody

What is Mono-methyl-HIST1H1E (K63) Antibody and what specific target does it detect?

Mono-methyl-HIST1H1E (K63) Antibody is a rabbit polyclonal antibody designed to recognize human histone H1.4 (HIST1H1E) proteins that are monomethylated specifically at the lysine 63 position . This antibody was developed using a synthesized peptide derived from human Histone H1.4 protein corresponding to amino acids 57-69 . The antibody detects this specific post-translational modification with high specificity, making it valuable for studying targeted histone modifications in chromatin regulation research.

Histone H1.4, encoded by the H1-4 gene (previously known as HIST1H1E), is a linker histone that plays important roles in higher-order chromatin structure and transcriptional regulation . The monomethylation at K63 represents one of multiple methylation states possible at lysine residues, which also include di- and tri-methylation, each potentially signaling different biological functions .

What applications are recommended for Mono-methyl-HIST1H1E (K63) Antibody in research settings?

The Mono-methyl-HIST1H1E (K63) Antibody has been validated for use in several research applications:

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection of monomethylated K63 H1.4 in cell or tissue lysates .

• Immunofluorescence/Immunocytochemistry (IF/ICC): For visualizing the subcellular localization of monomethylated K63 H1.4 within cells .

When designing experiments, researchers should note that optimal dilutions must be determined empirically for each experimental system and application . The antibody is supplied in unconjugated form, allowing flexibility in detection methods through secondary antibody selection . While not explicitly tested in the provided data, researchers might consider adapting protocols from related methylation-specific antibodies for applications such as chromatin immunoprecipitation (ChIP) or Western blotting to study the genomic distribution or expression levels of this modification.

How does histone monomethylation differ functionally from other lysine modifications?

Lysine residues in histones can undergo various post-translational modifications including methylation, acetylation, and ubiquitination, each with distinct biological implications:

• Monomethylation vs. Di/Trimethylation: Lysine residues can accept up to three methyl groups, with each methylation state potentially signaling different functions . For instance, in histone H3, monomethylation at K4 (H3K4me1) is primarily found at enhancers, while trimethylation (H3K4me3) marks active promoters .

• Methylation vs. Ubiquitination: While methylation of lysine residues like K63 in HIST1H1E may affect protein-protein interactions or chromatin structure, ubiquitination of lysines serves different functions. K63-linked polyubiquitination, for example, plays roles in DNA damage response, endocytosis, and signaling pathway activation rather than protein degradation .

• Regulatory Mechanisms: Monomethylation is catalyzed by specific protein lysine methyltransferases (PKMTs), and the modification can be removed by histone demethylases, allowing for dynamic regulation of chromatin structure and function .

The specific functions of monomethylation at K63 of HIST1H1E are still being investigated, but may relate to chromatin compaction, transcriptional regulation, or cellular responses to environmental stimuli.

How can Mono-methyl-HIST1H1E (K63) Antibody be integrated into studies of oxidative stress response?

Researchers investigating oxidative stress responses may find the Mono-methyl-HIST1H1E (K63) Antibody particularly valuable based on emerging connections between K63 modifications and stress responses:

• Monitoring Modification Changes: The antibody can be used to track changes in K63 monomethylation levels of HIST1H1E during oxidative stress exposure, potentially revealing new epigenetic responses .

• Combined Analysis with Ubiquitination: While K63-linked polyubiquitination increases during oxidative stress response , researchers could investigate whether K63 monomethylation of HIST1H1E exhibits coordinated or antagonistic changes, potentially indicating regulatory crosstalk between these modifications.

• Ribosomal Interaction Studies: Given that K63 ubiquitination targets ribosomal proteins during oxidative stress , researchers might use this antibody to investigate whether HIST1H1E monomethylation is associated with ribosome function or translation regulation under stress conditions.

Experimental approaches might include treating cells with hydrogen peroxide (H₂O₂) or other oxidative stressors, then using the antibody for immunofluorescence to visualize changes in nuclear distribution or for immunoprecipitation followed by mass spectrometry to identify interaction partners.

What methodological considerations are important when validating Mono-methyl-HIST1H1E (K63) Antibody specificity?

Validating antibody specificity is crucial for reliable research results. For Mono-methyl-HIST1H1E (K63) Antibody, consider these methodological approaches:

• Peptide Competition Assay: Pre-incubate the antibody with excess synthetic monomethylated K63 peptide before application in your experimental system. Loss of signal confirms specificity .

• Cross-reactivity Testing: Evaluate potential cross-reactivity with:

  • Unmethylated HIST1H1E

  • Di- or tri-methylated K63 HIST1H1E

  • Other methylated histones, particularly H1 variants

• Knockout/Knockdown Controls: Use HIST1H1E knockout/knockdown systems or cells treated with methyltransferase inhibitors to confirm signal dependence on the target modification.

• Modification-specific Western Blotting: If adapting for Western blot applications, confirm a single band at the expected molecular weight (approximately 21.9 kDa for H1.4) .

• Recombinant Protein Controls: Use recombinant HIST1H1E proteins with defined methylation states as positive and negative controls for calibration.

Document antibody lot number, dilution optimization experiments, and validation results thoroughly to ensure experimental reproducibility and reliability.

How might methylation of HIST1H1E K63 intersect with cancer biology and epigenetic dysregulation?

The potential role of HIST1H1E K63 monomethylation in cancer biology represents an important research avenue:

• Altered Methylation Patterns: Aberrant transcription mediated by methyltransferases has significant impacts on gene regulation and is linked to developmental disorders and cancer . Researchers could use the Mono-methyl-HIST1H1E (K63) Antibody to determine whether this specific modification is altered in cancer cells.

• Genomic Mutations Analysis: HIST1H1E is frequently altered in human cancers alongside other epigenetic regulators like EZH2, DNMT3A, and PTEN . The antibody could help characterize the functional consequences of these mutations on monomethylation levels.

• PIK3CA Pathway Interactions: Given that HIST1H1E alterations co-occur with PIK3CA mutations in overgrowth-related cancers , researchers might investigate the relationship between PI3K signaling and HIST1H1E K63 monomethylation.

• Therapeutic Response Monitoring: The antibody could be applied to determine whether cancer treatments affect K63 monomethylation levels, potentially revealing epigenetic mechanisms of drug response or resistance.

Experimental approaches might include comparative immunostaining of normal and cancerous tissues, ChIP-seq to map genomic distribution changes, or combining with transcriptomic analysis to correlate modification levels with gene expression changes in cancer progression.

What is the recommended protocol for using Mono-methyl-HIST1H1E (K63) Antibody in immunofluorescence studies?

The following protocol provides a starting point for immunofluorescence applications, though optimization for specific cell types and experimental conditions is essential:

Materials:

• Mono-methyl-HIST1H1E (K63) Antibody

• Appropriate secondary antibody (anti-rabbit IgG with desired fluorophore)

• 4% paraformaldehyde in PBS

• 0.1% Triton X-100 in PBS

• Blocking solution (3% BSA in PBS)

• DAPI or alternative nuclear counterstain

• Mounting medium

Procedure:

• Cell Preparation:

  • Culture cells on coverslips or chamber slides

  • Wash cells with PBS (2 × 5 minutes)

• Fixation and Permeabilization:

  • Fix cells with 4% paraformaldehyde (15 minutes, room temperature)

  • Wash with PBS (3 × 5 minutes)

  • Permeabilize with 0.1% Triton X-100 (10 minutes, room temperature)

  • Wash with PBS (3 × 5 minutes)

• Blocking and Antibody Incubation:

  • Block with 3% BSA (1 hour, room temperature)

  • Incubate with Mono-methyl-HIST1H1E (K63) Antibody diluted in blocking solution (start with 1:200 dilution, overnight at 4°C)

  • Wash with PBS (3 × 10 minutes)

  • Incubate with fluorophore-conjugated secondary antibody (1 hour, room temperature)

  • Wash with PBS (3 × 10 minutes)

• Counterstaining and Mounting:

  • Counterstain with DAPI (5 minutes)

  • Wash briefly with PBS

  • Mount with anti-fade mounting medium

• Imaging:

  • Visualize using confocal or fluorescence microscopy with appropriate filters

Troubleshooting Tips:

• If signal is weak, increase antibody concentration or incubation time

• For high background, increase blocking time or washing steps

• Always include a negative control (omitting primary antibody)

• Consider including a positive control tissue known to express monomethylated HIST1H1E K63

What are the most common technical challenges when working with Mono-methyl-HIST1H1E (K63) Antibody and how can they be addressed?

When working with Mono-methyl-HIST1H1E (K63) Antibody, researchers may encounter several technical challenges:

• Low Signal Intensity:

  • Cause: Insufficient antibody concentration, low target abundance, or suboptimal detection system

  • Solution: Titrate antibody (try 1:100 to 1:500 dilutions); use signal amplification methods like tyramide signal amplification; optimize fixation to preserve epitope

• Non-specific Background:

  • Cause: Insufficient blocking, excessive antibody concentration, or cross-reactivity

  • Solution: Increase blocking time/concentration; include 0.1-0.3% Tween-20 in wash buffers; use proper negative controls; perform peptide competition assay to confirm specificity

• Inconsistent Results Between Experiments:

  • Cause: Variations in sample preparation, antibody handling, or cellular conditions

  • Solution: Standardize protocols; prepare larger antibody aliquots to reduce freeze-thaw cycles; store at -20°C to maintain stability ; include internal controls in each experiment

• Epitope Masking:

  • Cause: Fixation may mask the methyl-K63 epitope

  • Solution: Test multiple fixation methods (paraformaldehyde, methanol, acetone); consider antigen retrieval methods adapted from histology protocols

• Variable Methylation Levels:

  • Cause: Methylation can be dynamic and affected by cell cycle or stress

  • Solution: Synchronize cells; standardize culture conditions; consider cellular stress factors that might alter methylation patterns

Maintaining proper antibody storage conditions (aliquoted at -20°C, avoiding repeated freeze-thaw cycles) is essential for preserving antibody performance over time .

How should researchers design appropriate controls for experiments using Mono-methyl-HIST1H1E (K63) Antibody?

Robust experimental design requires appropriate controls when using Mono-methyl-HIST1H1E (K63) Antibody:

Essential Controls:

• Negative Controls:

  • No Primary Antibody: Include samples processed identically but omitting the primary antibody to assess secondary antibody specificity

  • Isotype Control: Use non-specific rabbit IgG at the same concentration to evaluate non-specific binding

  • Blocking Peptide: Pre-incubate antibody with excess monomethylated K63 peptide to demonstrate binding specificity

• Positive Controls:

  • Known Positive Cell Type: Include a cell line or tissue known to express monomethylated HIST1H1E K63

  • Recombinant Standards: Use recombinant proteins with defined methylation states when available

• Validation Controls:

  • Methyltransferase Inhibitors: Treat cells with appropriate methyltransferase inhibitors to reduce the modification

  • Genetic Controls: When possible, use cells with CRISPR-mediated mutation of K63 to alanine or arginine

• Sample Processing Controls:

  • Technical Replicates: Process multiple samples to assess reproducibility

  • Loading Controls: For quantitative applications, normalize to appropriate loading controls

Control Experiments Table:

Documenting these controls thoroughly in publications strengthens the reliability and reproducibility of research findings.

How might Mono-methyl-HIST1H1E (K63) Antibody be utilized in studying connections between histone modifications and ubiquitination pathways?

The relationship between histone methylation and ubiquitination represents an exciting research frontier where Mono-methyl-HIST1H1E (K63) Antibody could provide valuable insights:

• Co-occurrence Analysis: Researchers can utilize the antibody in co-immunoprecipitation or sequential chromatin immunoprecipitation (ChIP-reChIP) experiments to determine whether K63 monomethylation and K63 ubiquitination co-occur or are mutually exclusive on the same histone molecules .

• Stress Response Studies: Given that K63-linked polyubiquitination increases during oxidative stress response , experiments could investigate whether K63 monomethylation of HIST1H1E changes in coordination with ubiquitination patterns during stress.

• Ribosomal Function Investigation: K63 polyubiquitination targets ribosomal proteins during stress and affects translation . Researchers could explore whether HIST1H1E K63 monomethylation occurs on ribosome-associated histones or influences translational processes.

• DNA Damage Response: Since K63-linked polyubiquitination plays roles in DNA damage response , the antibody could help determine if HIST1H1E K63 monomethylation participates in similar pathways through immunofluorescence co-localization studies with DNA damage markers.

• Enzymatic Cross-regulation: Investigate whether enzymes involved in K63 ubiquitination (like Rad6-Bre1) and those mediating K63 methylation interact or influence each other's activity.

These studies could reveal previously unknown crosstalk between these distinct post-translational modification pathways, potentially uncovering new regulatory mechanisms in cellular stress responses.

What considerations are important when adapting Mono-methyl-HIST1H1E (K63) Antibody for chromatin immunoprecipitation (ChIP) experiments?

While not explicitly validated for ChIP in the provided information, researchers may adapt the Mono-methyl-HIST1H1E (K63) Antibody for this application with careful optimization:

• Antibody Qualification:

  • Perform preliminary IP experiments to verify the antibody can effectively immunoprecipitate its target

  • Validate antibody specificity through Western blotting of input and immunoprecipitated material

• Crosslinking Optimization:

  • Test different formaldehyde concentrations (typically 0.75-1.5%) and crosslinking times

  • Consider dual crosslinking (formaldehyde plus ethylene glycol bis-succinimidyl succinate) for improved histone modification detection

• Chromatin Fragmentation:

  • Optimize sonication or enzymatic digestion to yield fragments of 200-500 bp

  • Verify fragmentation efficiency by agarose gel electrophoresis

• Antibody Amount Optimization:

  • Titrate antibody amounts (typically 2-10 μg per IP reaction)

  • Include appropriate IgG control at matching concentration

• Washing Stringency:

  • Adjust salt concentration in wash buffers to optimize signal-to-noise ratio

  • Consider including detergents like Triton X-100 or SDS at appropriate concentrations

• ChIP-seq Considerations:

  • For genome-wide studies, ensure sufficient sequencing depth (typically >20 million mapped reads)

  • Include input controls for normalization

  • Consider spike-in controls for quantitative comparisons between conditions

• Validation Approaches:

  • Confirm enrichment at expected genomic regions by qPCR before proceeding to sequencing

  • Validate findings with orthogonal approaches (e.g., CUT&RUN or CUT&Tag)

The purification method of this antibody (antigen affinity chromatography) suggests it may have sufficient specificity for ChIP applications when properly optimized.

How does research on HIST1H1E K63 monomethylation relate to broader epigenetic regulation in development and disease?

Research utilizing Mono-methyl-HIST1H1E (K63) Antibody could contribute to our understanding of epigenetic regulation in several ways:

• Histone Code Expansion: The detection of K63 monomethylation adds to our understanding of the "histone code" – the complex language of histone modifications that regulate chromatin structure and function . Researchers can map this modification genome-wide to determine its relationship with transcriptionally active or repressed regions.

• Cancer Connections: HIST1H1E is frequently altered in human cancers alongside other epigenetic regulators including EZH2, DNMT3A, and PTEN . Studies examining changes in K63 monomethylation patterns across cancer progression could reveal new biomarkers or therapeutic targets.

• Developmental Regulation: Histone modifications play crucial roles in developmental gene regulation. The antibody could help characterize changes in K63 monomethylation during cellular differentiation, potentially revealing stage-specific epigenetic signatures.

• PIK3CA Pathway Interactions: Given HIST1H1E alterations co-occur with PIK3CA mutations in overgrowth-related disorders , research could investigate connections between signaling pathways and histone modifications.

• Therapeutic Implications: Understanding the enzymes responsible for K63 monomethylation could identify potential targets for epigenetic therapies, similar to existing methyltransferase inhibitors for cancer treatment.

This research area represents an intersection between histone biology, signaling pathway regulation, and disease processes, with implications for both basic science understanding and translational medicine.

What technological advances might enhance research using Mono-methyl-HIST1H1E (K63) Antibody?

Emerging technologies could significantly expand applications of Mono-methyl-HIST1H1E (K63) Antibody:

• CUT&RUN and CUT&Tag:

  • These antibody-directed chromatin profiling techniques offer higher signal-to-noise ratios than traditional ChIP

  • Require less starting material (as few as 1,000 cells)

  • Could provide more precise genomic mapping of K63 monomethylated HIST1H1E

• Single-Cell Epigenomics:

  • Adapting the antibody for single-cell techniques could reveal cell-to-cell variation in K63 monomethylation

  • Single-cell CUT&Tag or antibody-based imaging methods could track modification dynamics in heterogeneous cell populations

• Proximity Labeling Proteomics:

  • Coupling the antibody with BioID or APEX2 proximity labeling systems

  • Could identify proteins interacting with K63 monomethylated HIST1H1E

  • Would reveal functional protein complexes associated with this modification

• Live-Cell Imaging:

  • Development of recombinant modification-specific intrabodies

  • Would enable tracking K63 monomethylation dynamics in living cells

  • Could reveal real-time changes during cellular processes

• Mass Spectrometry Integration:

  • Combining antibody-based enrichment with targeted mass spectrometry

  • Would allow quantitative analysis of K63 monomethylation and co-occurring modifications

  • Similar to methods used for K63 polyubiquitin linkage analysis

These technological advancements would provide more comprehensive insights into the dynamic regulation and functional significance of HIST1H1E K63 monomethylation in various biological contexts.

How might HIST1H1E K63 monomethylation interact with oxidative stress response mechanisms?

Given the connections between K63 modifications and oxidative stress response , this research area presents intriguing possibilities:

• Temporal Dynamics:

  • Researchers could use the antibody to track temporal changes in K63 monomethylation following oxidative stress

  • Compare with the rapid K63 polyubiquitination response (occurring within 5 minutes of H₂O₂ treatment)

  • Determine whether methylation changes precede, coincide with, or follow ubiquitination

• Ribosomal Association:

  • Since K63 polyubiquitination targets ribosomal proteins during stress , investigate whether HIST1H1E K63 monomethylation occurs on nucleosomes associated with ribosomal genes

  • Examine potential role in regulating ribosomal gene expression under stress conditions

• Enzyme Regulation:

  • Study whether oxidative stress affects the activity of methyltransferases targeting HIST1H1E K63

  • Investigate if enzymes like Rad6-Bre1, involved in K63 polyubiquitination during stress , influence K63 monomethylation

• Redox Sensitivity:

  • Determine if HIST1H1E K63 monomethylation is sensitive to cellular redox state

  • Examine whether it serves as part of a redox-sensing mechanism affecting chromatin structure

• Transcriptional Response:

  • Map genomic locations of K63 monomethylated HIST1H1E before and after oxidative stress

  • Correlate with stress-responsive gene expression changes

  • Determine if the modification marks stress-responsive regulatory elements

This research could reveal previously unknown epigenetic mechanisms involved in cellular adaptation to oxidative stress, with potential implications for aging, cancer, and neurodegenerative diseases where oxidative stress plays significant roles.

What are the potential applications of Mono-methyl-HIST1H1E (K63) Antibody in neurodegenerative disease research?

Epigenetic dysregulation is increasingly recognized in neurodegenerative disorders, suggesting several applications for this antibody:

• Oxidative Stress Connection:

  • Neurodegenerative diseases often feature oxidative stress and protein modification changes

  • The antibody could help track K63 monomethylation alterations in disease models

  • May reveal connections to known oxidative stress response pathways involving K63 modifications

• Brain Region-Specific Patterns:

  • Compare K63 monomethylation patterns across brain regions differentially affected in diseases like Alzheimer's or Parkinson's

  • Analyze changes in vulnerable neuronal populations versus resistant ones

• Age-Related Changes:

  • Investigate whether K63 monomethylation of HIST1H1E changes with aging

  • Correlate with known age-related epigenetic drift patterns

  • Examine potential connections to cellular senescence

• Therapeutic Response Monitoring:

  • Track K63 monomethylation changes in response to neuroprotective interventions

  • Determine if epigenetic-targeting drugs affect this modification

  • Potential development as a biomarker for treatment efficacy

• Protein Aggregation Interactions:

  • Examine relationships between K63 monomethylation and protein aggregation processes

  • Investigate potential connections to protein quality control pathways that involve K63 polyubiquitination

These applications could help elucidate the role of histone modifications in neurodegeneration and potentially identify new therapeutic strategies targeting epigenetic mechanisms.