Mono-methyl-HIST1H4A (K59) Antibody

What is HIST1H4A and what role does K59 methylation play in epigenetic regulation?

HIST1H4A is the gene encoding Histone H4, a core component of the nucleosome. Nucleosomes wrap and compact DNA into chromatin, limiting DNA accessibility to cellular machineries that require DNA as a template. Histones play a central role in transcription regulation, DNA repair, DNA replication, and chromosomal stability . The mono-methylation at lysine 59 (K59) of Histone H4 is one of the many post-translational modifications that make up the "histone code" which regulates DNA accessibility and chromatin remodeling. This specific modification contributes to the complex system of epigenetic regulation that determines which genes are accessible for transcription.

How does Mono-methyl-HIST1H4A (K59) differ from other histone modifications?

Mono-methyl-HIST1H4A (K59) is distinct from other histone modifications in terms of its location, function, and regulatory impact. Unlike the well-characterized H3K4 methylation which is associated with active transcription , K59 methylation on Histone H4 operates within its own regulatory context. The specificity of this modification allows researchers to examine unique aspects of chromatin regulation that may not be captured by studying more common histone marks. Different histone modifications create a combinatorial code that precisely controls gene expression patterns, with K59 methylation contributing to this intricate regulatory network.

What biological processes are regulated by Mono-methyl-HIST1H4A (K59)?

The mono-methylation of Histone H4 at lysine 59 influences various biological processes through its impact on chromatin structure and transcriptional regulation. This modification affects DNA accessibility, which in turn regulates processes including gene expression, DNA replication, and cellular differentiation . Understanding how this specific modification contributes to these processes provides insights into fundamental mechanisms of epigenetic regulation that govern cellular function and development.

What are the validated applications for Mono-methyl-HIST1H4A (K59) antibody?

According to the provided information, the Mono-methyl-HIST1H4A (K59) antibody has been validated for several key applications in epigenetic research:

These applications enable researchers to detect and quantify K59 methylation in various experimental contexts .

What protocols are recommended for optimal western blot detection of Mono-methyl-HIST1H4A (K59)?

For optimal western blot detection of Mono-methyl-HIST1H4A (K59), researchers should follow these methodological guidelines:

• Sample preparation: Extract histones using an acid extraction protocol to enrich for basic proteins.

• Gel selection: Use high percentage (15-18%) SDS-PAGE gels to properly resolve the low molecular weight (12 kDa) histone proteins.

• Transfer conditions: Implement methanol-containing transfer buffer and PVDF membranes for optimal protein transfer.

• Blocking: Block with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature.

• Primary antibody: Dilute the Mono-methyl-HIST1H4A (K59) antibody at 1:100-1:1000 in blocking buffer and incubate overnight at 4°C .

• Secondary antibody: Use anti-rabbit IgG at recommended dilutions (typically 1:50000).

• Detection: Utilize enhanced chemiluminescence (ECL) for visualization.

Validated positive controls include HeLa and 293 whole cell lysates, which consistently show the expected 12 kDa band corresponding to histone H4 .

How should immunofluorescence experiments be designed for optimal detection of this histone modification?

For immunofluorescence experiments targeting Mono-methyl-HIST1H4A (K59), researchers should implement this detailed protocol:

• Cell preparation: Culture cells on coverslips to 70-80% confluence.

• Fixation: Fix cells with 4% formaldehyde in PBS for 10-15 minutes at room temperature.

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

• Blocking: Block with 10% normal goat serum for 1 hour to reduce non-specific binding.

• Primary antibody: Apply Mono-methyl-HIST1H4A (K59) antibody at a dilution of 1:1-1:10 and incubate overnight at 4°C.

• Secondary antibody: Use fluorophore-conjugated secondary antibodies (e.g., Alexa Fluor 488-conjugated anti-rabbit IgG).

• Nuclear counterstain: Apply DAPI for nuclear visualization.

• Mounting: Mount slides with anti-fade mounting medium.

This methodology has been validated in HeLa cells and provides clear nuclear staining patterns that reflect the distribution of this histone modification .

How does the specificity of Mono-methyl-HIST1H4A (K59) antibody compare to antibodies for other histone modifications?

The specificity of the Mono-methyl-HIST1H4A (K59) antibody is a critical consideration in epigenetic research. Antibody specificity for histone modifications is determined by:

• Epitope recognition: The antibody specifically recognizes the peptide sequence surrounding the mono-methylated lysine at position 59 of Histone H4.

• Cross-reactivity assessment: Rigorous testing ensures minimal cross-reactivity with other histone modifications.

• Validation across applications: The antibody demonstrates consistent results across multiple experimental platforms (ELISA, WB, IF) .

Unlike antibodies for more common modifications like H3K4me1 , which have been extensively characterized across multiple species and experimental systems, antibodies for specialized modifications like H4K59me1 require particular attention to validation parameters. Researchers should always perform appropriate control experiments to confirm specificity in their particular experimental context.

What are the optimal conditions for studying dynamic changes in K59 methylation during cellular processes?

To effectively study the dynamics of K59 methylation during cellular processes, researchers should consider these methodological approaches:

• Time-course experiments: Sample collection at multiple time points following stimulation or treatment.

• Synchronization protocols: Use cell cycle synchronization methods to study cell cycle-dependent changes.

• ChIP-sequencing approaches: Apply chromatin immunoprecipitation followed by sequencing to map genome-wide distribution.

• Live-cell imaging: Consider developing fluorescent reporters for real-time monitoring when feasible.

• Quantitative western blotting: Implement standardized protocols with proper loading controls.

• Mass spectrometry validation: Confirm changes in methylation levels through mass spectrometry-based approaches.

For all these approaches, the Mono-methyl-HIST1H4A (K59) antibody should be used at experimentally determined optimal concentrations, typically 1:100-1:1000 for western blotting or 1:1-1:10 for immunofluorescence studies .

How can researchers differentiate between mono-, di-, and tri-methylation of K59 in experimental settings?

Distinguishing between different methylation states at K59 requires careful experimental design and appropriate controls:

• Antibody selection: Use highly specific antibodies that discriminate between mono-, di-, and tri-methylation states. The Mono-methyl-HIST1H4A (K59) antibody is specifically designed to recognize only the monomethylated form.

• Peptide competition assays: Conduct peptide competition assays using synthetic peptides with defined methylation states to confirm antibody specificity.

• Mass spectrometry: Implement mass spectrometry-based approaches to unambiguously identify and quantify different methylation states.

• Methylation-specific mutants: When possible, use cell lines expressing histone mutants that cannot be methylated at specific sites as negative controls.

• Known samples: Include samples with verified methylation states as positive and negative controls.

These approaches help ensure that the observed signals truly represent the specific methylation state of interest rather than cross-reactivity with other modifications.

What are common problems when using Mono-methyl-HIST1H4A (K59) antibody and how can they be resolved?

Researchers frequently encounter these challenges when working with the Mono-methyl-HIST1H4A (K59) antibody:

Proper storage of the antibody (50% Glycerol, 0.01M PBS, pH 7.4 with 0.03% Proclin 300 as preservative) at recommended conditions helps maintain its activity and specificity over time .

How can researchers validate the specificity of the Mono-methyl-HIST1H4A (K59) antibody in their experimental system?

To validate the specificity of the Mono-methyl-HIST1H4A (K59) antibody in a particular experimental system, researchers should implement these methodological approaches:

• Peptide competition assays: Pre-incubate the antibody with the immunizing peptide to confirm that this blocks specific binding.

• Knockdown/knockout validation: Use cells with reduced or eliminated expression of the histone methyltransferase responsible for K59 methylation.

• Histone demethylase overexpression: Overexpress the relevant demethylase to reduce K59 methylation levels.

• Dot blot analysis: Test antibody against synthetic peptides containing different histone modifications.

• Multiple detection methods: Confirm findings using multiple techniques (WB, IF, ELISA) to ensure consistency.

• Known positive controls: Include samples with established K59 methylation, such as HeLa or 293 cell lysates .

These validation steps ensure that experimental observations genuinely reflect the intended histone modification rather than non-specific interactions.

What extraction methods provide optimal results for detecting Mono-methyl-HIST1H4A (K59)?

Obtaining high-quality histone preparations is crucial for detecting Mono-methyl-HIST1H4A (K59). These extraction methods provide optimal results:

• Acid extraction protocol:

  • Lyse cells in hypotonic buffer

  • Extract histones with 0.2N HCl

  • Neutralize with NaOH

  • Quantify protein concentration

• Histone purification protocol:

  • Isolate nuclei through differential centrifugation

  • Extract histones with high salt buffer

  • Purify histones through size exclusion or ion exchange chromatography

• Direct lysis method for western blotting:

  • Lyse cells directly in SDS sample buffer

  • Sonicate to shear genomic DNA

  • Heat samples at 95°C for 5 minutes

These methods ensure preservation of histone modifications while removing potentially interfering proteins and cellular components, yielding samples ideally suited for antibody-based detection of specific modifications like K59 methylation.

How does Mono-methyl-HIST1H4A (K59) function within the broader context of the histone code?

The mono-methylation of Histone H4 at lysine 59 functions as part of the complex histone code that regulates chromatin structure and gene expression. This modification works in concert with other histone modifications to create specific combinatorial patterns that determine the functional state of chromatin regions . Within this context, K59 methylation likely serves as a specific regulatory mark that influences the recruitment of effector proteins that recognize this modification.

The histone code hypothesis suggests that these modifications are "read" by specific proteins that then influence downstream processes such as transcription, replication, or DNA repair. Understanding how K59 methylation fits into this broader regulatory framework requires integration of data from multiple histone modifications and their associated protein complexes.

What chromatin remodeling complexes interact with mono-methylated K59 on Histone H4?

While the specific chromatin remodeling complexes that interact with mono-methylated K59 on Histone H4 are not explicitly stated in the provided search results, researchers can investigate this question using these methodological approaches:

• Affinity purification using modified histone peptides as bait

• Mass spectrometry analysis of proteins that co-immunoprecipitate with mono-methylated H4K59

• Protein domain arrays to identify specific protein domains that recognize this modification

• In vitro binding assays with recombinant reader domain proteins

• Proximity labeling approaches in cells to identify proteins in close proximity to this modification

These experimental strategies can reveal the protein complexes that specifically recognize mono-methylated K59, providing insight into how this modification influences chromatin structure and function.

How can researchers integrate Mono-methyl-HIST1H4A (K59) data with other epigenetic datasets?

Integrating data on Mono-methyl-HIST1H4A (K59) with other epigenetic information requires sophisticated bioinformatic approaches:

• Genome-wide correlation analysis: Compare ChIP-seq profiles of H4K59me1 with other histone modifications to identify co-occurrence patterns.

• Motif analysis: Identify DNA sequence motifs associated with H4K59me1 enrichment to discover potential regulatory connections.

• Integration with transcriptomic data: Correlate H4K59me1 levels with gene expression data to understand functional impacts.

• Multi-omics integration: Combine H4K59me1 profiles with DNA methylation, chromatin accessibility, and 3D genome structure data.

• Network analysis: Build interaction networks that include enzymes regulating H4K59 methylation and their connections to other epigenetic modulators.

These approaches permit researchers to place H4K59 methylation within the broader context of epigenetic regulation, revealing its specific role in chromatin structure and gene expression control.

What emerging technologies might enhance the study of Mono-methyl-HIST1H4A (K59)?

Emerging technologies that could significantly advance the study of Mono-methyl-HIST1H4A (K59) include:

• Single-cell epigenomics: Technologies that enable mapping of H4K59me1 at single-cell resolution to reveal cell-to-cell heterogeneity.

• CRISPR-based epigenome editing: Targeted modification of K59 methylation at specific genomic loci to assess functional consequences.

• Live-cell imaging of histone modifications: Development of specific sensors for real-time visualization of dynamic changes in K59 methylation.

• Mass spectrometry innovations: Improved sensitivity and throughput for quantitative analysis of histone modifications.

• Spatial epigenomics: Methods for mapping H4K59me1 within the 3D nuclear architecture.

These technological advances promise to provide unprecedented insights into the spatial and temporal dynamics of K59 methylation and its functional significance in various biological contexts.

How can computational models help predict the functional impact of K59 methylation patterns?

Computational models offer powerful approaches for understanding the functional significance of K59 methylation:

• Machine learning algorithms: Train models on integrated epigenomic datasets to predict functional states associated with K59 methylation patterns.

• Molecular dynamics simulations: Model how K59 methylation affects nucleosome structure and dynamics.

• Network analysis: Construct regulatory networks involving K59 methylation to predict its impact on gene expression.

• Statistical modeling: Develop statistical frameworks to identify significant associations between K59 methylation and phenotypic outcomes.

• Comparative genomics: Analyze conservation of K59 methylation patterns across species to infer functional importance.

These computational approaches complement experimental studies by generating testable hypotheses about the functional significance of K59 methylation in different biological contexts.

What are the most promising therapeutic applications related to modulating K59 methylation?

While the therapeutic applications of modulating K59 methylation are not directly addressed in the provided search results, several promising research directions can be identified:

• Cancer epigenetics: Investigate altered patterns of K59 methylation in cancer cells and potential therapeutic interventions targeting the enzymes responsible.

• Neurodegenerative disorders: Explore connections between K59 methylation and neuronal gene expression patterns relevant to neurodegeneration.

• Inflammatory diseases: Study how K59 methylation influences the expression of inflammatory genes and potential anti-inflammatory interventions.

• Developmental disorders: Examine the role of K59 methylation in developmental gene regulation and potential therapeutic approaches.

• Aging: Investigate changes in K59 methylation during aging and interventions that might restore youthful methylation patterns.

These research directions represent potential pathways through which understanding of K59 methylation could be translated into novel therapeutic approaches for various diseases.