Mono-methyl-HIST1H3A (K9) Recombinant Monoclonal Antibody

What is Mono-methyl-HIST1H3A (K9) and why is it significant in epigenetic research?

Mono-methyl-HIST1H3A (K9) refers to the monomethylation of lysine 9 on histone H3, a core component of nucleosomes. This epigenetic modification plays a central role in transcription regulation, DNA repair, DNA replication, and chromosomal stability. Histones are subject to complex post-translational modifications, collectively known as the histone code, which regulate DNA accessibility to cellular machinery . Mono-methylation at K9 specifically is linked to transcriptional repression mechanisms, making it a critical target for researchers studying chromatin dynamics and gene regulation.

How does a Mono-methyl-HIST1H3A (K9) antibody differ from antibodies targeting other histone H3 methylation states?

While all targeting histone H3 modifications, these antibodies recognize distinct epitopes:

Specificity testing confirms that mono-methyl K9 antibodies do not cross-react with di-methyl or tri-methyl K9 modifications, nor with modifications at other lysine residues such as K27 or K4 . This specificity is critical for accurate interpretation of experimental results.

What are the key specifications of commercially available Mono-methyl-HIST1H3A (K9) Recombinant Monoclonal Antibodies?

Recombinant monoclonal antibodies targeting H3K9me1 typically have the following specifications:

• Host/Isotype: Rabbit IgG

• Clonality: Monoclonal (recombinant)

• Applications: Western Blot (WB), Immunofluorescence (IF), Immunocytochemistry (ICC), ChIP, ELISA

• Reactivity: Human, mouse, rat

• Molecular Weight: Typically detected at 15-18 kDa (predicted 15 kDa)

• Storage Conditions: -20°C in PBS with 0.02% sodium azide and 50% glycerol (pH 7.3)

What are the validated applications for Mono-methyl-HIST1H3A (K9) antibodies and their optimal working dilutions?

Based on validation data from manufacturers, these antibodies perform reliably in multiple applications:

Experimental validation has confirmed reactivity in multiple cell lines including HeLa, HEK-293, THP-1, K-562, Jurkat, NIH/3T3, C2C12, HSC-T6, and PC-12 cells .

What is the optimal Western blot protocol for detecting Mono-methyl-HIST1H3A (K9)?

For optimal Western blot results:

• Sample Preparation: Extract nuclear proteins or isolate histones using acid extraction.

• Gel Electrophoresis: Run 10-30 μg of nuclear lysate on a 5-20% SDS-PAGE gel at 70V (stacking)/90V (resolving) for 2-3 hours .

• Transfer: Transfer proteins to nitrocellulose membrane at 150 mA for 50-90 minutes .

• Blocking: Block membrane with 5% non-fat milk in TBS for 1.5 hours at room temperature .

• Primary Antibody: Incubate with anti-H3K9me1 antibody at 1:500-1:5000 dilution overnight at 4°C .

• Washing: Wash membrane with TBS-0.1% Tween 3 times, 5 minutes each .

• Secondary Antibody: Incubate with HRP-conjugated anti-rabbit IgG at 1:5000-1:10000 for 1-1.5 hours at room temperature .

• Detection: Develop using enhanced chemiluminescence (ECL) detection system .

The expected band for mono-methyl histone H3 (K9) appears at approximately 15-18 kDa .

What protocols yield optimal results for immunofluorescence detection of H3K9me1?

For successful IF/ICC staining:

• Fixation: Fix cells with either 4% paraformaldehyde (5-10 min) or 100% methanol (5 min) .

• Permeabilization: Permeabilize with 0.05-0.3% Triton X-100 in PBS or TBS .

• Blocking: Block with 2-10% normal goat serum and/or 1% BSA in PBS for 30-60 minutes .

• Primary Antibody: Incubate with anti-H3K9me1 antibody at 1:100-1:800 dilution for 1 hour at room temperature or overnight at 4°C .

• Secondary Antibody: Incubate with fluorophore-conjugated anti-rabbit IgG (e.g., Alexa Fluor 488) at 1:200-1:1000 dilution for 30-60 minutes at room temperature .

• Counterstaining: Counterstain nuclei with DAPI and cell membranes with WGA if desired .

• Visualization: Observe using a fluorescence microscope with appropriate filter sets .

H3K9me1 typically shows nuclear localization with potential enrichment in specific nuclear domains.

What are common challenges when working with Mono-methyl-HIST1H3A (K9) antibodies and how can they be addressed?

Peptide competition assays demonstrate that H3K9me1 antibodies can be successfully blocked by the immunizing peptide (mono-methyl K9) but not by peptides containing di-methyl K9, tri-methyl K9, or other modified residues .

How can I verify the specificity of a Mono-methyl-HIST1H3A (K9) antibody?

To confirm antibody specificity:

• Peptide Competition: Perform Western blot with antibody pre-incubated with mono-methyl K9 peptide versus other methylated states. A specific antibody will be blocked only by the mono-methyl K9 peptide but not by di-methyl K9, tri-methyl K9, mono-methyl K27, or unmodified peptides .

• Knockout/Knockdown Controls: Test antibody reactivity in cells with knockdown of enzymes responsible for H3K9 monomethylation (e.g., G9a). Western blot analysis shows reduced H3K9me1 signal in G9a siRNA-treated cells compared to untreated controls .

• Cross-Reactivity Testing: Examine reactivity against a panel of modified histone peptides using dot blots or ELISA to confirm specific recognition of H3K9me1.

• Application-Specific Validation: For ChIP applications, validate enrichment at known H3K9me1-marked regions versus negative control regions.

How can Mono-methyl-HIST1H3A (K9) antibodies be used for ChIP-seq applications?

For successful ChIP-seq experiments:

• Crosslinking: Fix cells with 1% formaldehyde for 10 minutes at room temperature.

• Chromatin Preparation: Lyse cells, isolate nuclei, and sonicate to generate 200-500 bp DNA fragments.

• Immunoprecipitation: Incubate chromatin with 2-5 μg anti-H3K9me1 antibody overnight at 4°C.

• Bead Capture: Add protein A/G magnetic beads, incubate 2-4 hours at 4°C.

• Washing: Perform stringent washes to remove non-specific binding.

• Elution and Reversal: Elute bound chromatin and reverse crosslinks.

• Library Preparation: Purify DNA and prepare sequencing libraries.

• Data Analysis: Align reads, call peaks, and analyze genomic distribution.

ChIP-grade antibodies against H3K9me1 have been validated in multiple cell types and show enrichment at specific genomic regions associated with transcriptional regulation .

What experimental designs can reveal functional relationships between H3K9me1 and other chromatin modifications?

To investigate functional relationships:

• Sequential ChIP (Re-ChIP): Perform successive immunoprecipitations with H3K9me1 antibody followed by antibodies against other modifications to identify co-occurrence.

• Integrated Multi-Omics: Combine ChIP-seq (H3K9me1), RNA-seq, ATAC-seq, and DNA methylation analysis from the same samples to correlate modifications with gene expression and chromatin accessibility.

• Perturbation Studies: Analyze H3K9me1 distribution following manipulation of writer/eraser enzymes or treatment with epigenetic inhibitors.

• Time-Course Experiments: Track changes in H3K9me1 distribution during cellular differentiation, response to stimuli, or cell cycle progression.

• Single-Cell Approaches: Combine with single-cell technologies to assess heterogeneity in H3K9me1 distribution across cell populations.

How should ChIP-seq data generated with Mono-methyl-HIST1H3A (K9) antibodies be analyzed?

For robust ChIP-seq data analysis:

• Quality Control: Assess sequencing quality, mapping rates, library complexity, and signal-to-noise ratios.

• Peak Calling: Use appropriate algorithms (e.g., MACS2) with parameters optimized for histone modifications.

• Genomic Distribution: Analyze H3K9me1 enrichment relative to genomic features (promoters, enhancers, gene bodies).

• Integration: Correlate H3K9me1 distribution with other histone marks, transcription factor binding, and gene expression.

• Differential Binding Analysis: Compare H3K9me1 patterns between experimental conditions.

• Motif Analysis: Identify DNA sequence motifs enriched in H3K9me1-marked regions.

• Pathway Analysis: Perform gene ontology and pathway enrichment for genes associated with H3K9me1 marks.

• Visualization: Create genome browser tracks and heatmaps to present spatial relationships.

What are the key considerations when interpreting Western blot data for histone modifications?

When analyzing Western blot results:

• Appropriate Controls: Include total histone H3 as a loading control rather than typical housekeeping genes.

• Molecular Weight Verification: Confirm the band appears at the expected size (15-18 kDa for H3) .

• Quantification Method: Use densitometry to quantify signal intensity, normalizing to total H3.

• Multiple Biological Replicates: Perform at least three independent experiments for statistical validity.

• Relative Quantification: Compare the ratio of modified to total histones rather than absolute values.

• Sample Preparation Effects: Be aware that extraction methods can affect retention of histone modifications.

• Antibody Specificity: Consider possible cross-reactivity with similar modifications (validated by peptide competition assays).

How can Mono-methyl-HIST1H3A (K9) antibodies be integrated with cutting-edge epigenomic technologies?

Innovative applications include:

• CUT&RUN/CUT&Tag: These techniques offer higher signal-to-noise ratios than traditional ChIP and require less starting material, making them valuable for rare cell populations.

• Single-Cell Epigenomics: Adaptation of H3K9me1 antibodies for single-cell ChIP-seq or CUT&Tag enables analysis of cellular heterogeneity.

• Spatial Epigenomics: Combining immunofluorescence with in situ sequencing to map H3K9me1 distribution in tissue context.

• Live-Cell Imaging: Development of recombinant antibody fragments for real-time tracking of H3K9me1 dynamics.

• CRISPR Epigenome Editing: Using H3K9me1 antibodies to validate targeted methylation/demethylation by CRISPR-based tools.

• Proteomics Integration: Combining ChIP with mass spectrometry to identify proteins associating with H3K9me1-marked chromatin.

What are the current gaps in understanding H3K9me1 function that could be addressed with improved antibody-based approaches?

Key research questions include:

• Cell-Type Specificity: How does H3K9me1 distribution vary across different cell types and tissues?

• Temporal Dynamics: What are the kinetics of H3K9me1 deposition and removal during cellular processes?

• Reader Proteins: Which proteins specifically recognize H3K9me1 and how do they function?

• Disease Associations: How are H3K9me1 patterns altered in disease states?

• Evolutionary Conservation: How conserved are H3K9me1 patterns across species?

• Crosstalk with Other Modifications: How does H3K9me1 interact with other histone marks and DNA modifications?

• Functional Consequences: What is the direct impact of H3K9me1 gain or loss on chromatin structure and gene expression?