HIST1H3A (Ab-17) Antibody

What is HIST1H3A and what specific modification does the Ab-17 antibody recognize?

HIST1H3A is one of several genes encoding histone H3.1, a core component of nucleosomes that wrap and compact DNA into chromatin. Histone H3 plays a central role in transcription regulation, DNA repair, DNA replication, and chromosomal stability through post-translational modifications that constitute the "histone code" .

The Ab-17 antibody specifically recognizes histone H3 with asymmetric dimethylation at arginine 17 (H3R17me2a). This particular modification is catalyzed primarily by the enzyme CARM1 (Coactivator-Associated Arginine Methyltransferase 1) . Importantly, this antibody demonstrates high specificity for the asymmetrically dimethylated form of R17 and does not cross-react with unmethylated histone H3, symmetrically dimethylated R17, or methylation at other arginine residues .

What experimental applications is the HIST1H3A (Ab-17) antibody validated for?

The antibody has been validated for multiple experimental applications:

This antibody recognizes synthetic peptide, human, and synthetic peptide-human samples, and has been cited in 59 publications, demonstrating its reliability in research settings .

How can I validate the specificity of the HIST1H3A (Ab-17) antibody?

Validating antibody specificity is critical for ensuring reliable experimental results. For the HIST1H3A (Ab-17) antibody, several approaches can be employed:

• Peptide Competition Assays: Perform antibody pre-absorption with unmethylated H3 peptide, R17 methylated H3 peptide, and other methylated peptides (e.g., R3 methylated H4) to confirm specificity .

• Dot Blot Analysis: Conduct dot blot using unmodified peptide, mono-methylated R17 peptide, asymmetrically dimethylated R17 peptide, and symmetrically dimethylated R17 peptide. The Ab-17 antibody should only recognize the asymmetrically dimethylated R17 peptide .

• Western Blot with Controls: Use recombinant unmethylated H3 alongside CARM1-methylated H3 as positive and negative controls, respectively .

• Methyltransferase Knockdown: In cell-based experiments, knock down CARM1 (the enzyme responsible for R17 methylation) and confirm reduced antibody signal.

Research indicates that all batches of this antibody are tested in Peptide Array against peptides with different Histone H3 modifications, with results confirming strong binding to Histone H3 - asymmetric di methyl R17 peptide (ab16935) .

What are the recommended experimental conditions for Western blotting with this antibody?

For optimal Western blot results with the HIST1H3A (Ab-17) antibody, the following conditions are recommended:

• Sample Preparation: Use histone preparations or nuclear lysates (e.g., HeLa Histone Preparation Nuclear Lysate at 2.5 μg/mL) .

• Antibody Dilution: Use the primary antibody at 1 μg/mL concentration .

• Secondary Antibody: Goat Anti-Rabbit IgG H&L (HRP) preadsorbed at 1/5000 dilution .

• Detection Method: ECL (Enhanced Chemiluminescence) technique .

• Reducing Conditions: Perform the electrophoresis under reducing conditions .

• Expected Results:

  • Predicted band size: 15 kDa

  • Observed band sizes: 17 kDa, 55 kDa, 60 kDa, 90 kDa

  • Recommended exposure time: 2 minutes

When interpreting results, note that the primary band corresponding to methylated histone H3 may appear at approximately 17 kDa rather than the predicted 15 kDa molecular weight, which is a common observation for histone proteins due to their post-translational modifications .

What is the biological significance of H3R17 methylation and how can this antibody help elucidate its function?

H3R17 methylation plays a crucial role in gene activation. This post-translational modification is catalyzed by CARM1 and is specifically associated with transcriptionally active chromatin states . Research using the Ab-17 antibody has revealed important insights into this modification's biological significance:

• Transcriptional Activation: Chromatin immunoprecipitation (ChIP) analyses have demonstrated that R17 methylation on histone H3 is dramatically upregulated when estrogen receptor-regulated genes (such as pS2) are activated .

• CARM1 Recruitment: The appearance of methylated R17 coincides with CARM1 association with histones on activated genes, providing direct evidence that CARM1-mediated methylation takes place in vivo during the active transcriptional state .

• Epigenetic Signaling: H3R17 methylation appears to be part of a cooperative network of histone modifications that work together to regulate transcription, potentially involving a cross-talk between acetylation and arginine methylation .

Researchers can use the Ab-17 antibody in ChIP experiments to identify genomic regions where this modification occurs, helping to map the epigenetic landscape associated with active transcription. This can provide insights into gene regulatory mechanisms in various biological contexts, including development, differentiation, and disease states.

How can I effectively use the HIST1H3A (Ab-17) antibody in chromatin immunoprecipitation (ChIP) experiments?

For successful ChIP experiments with the HIST1H3A (Ab-17) antibody, consider the following methodological recommendations:

• Crosslinking Optimization: Use 1% formaldehyde for 10 minutes at room temperature for effective DNA-protein crosslinking.

• Chromatin Preparation: Sonicate chromatin to fragments of approximately 200-500 bp for optimal immunoprecipitation.

• Antibody Amount: Use 2-5 μg of antibody per ChIP reaction, depending on the cell type and abundance of the modification.

• Controls:

  • Include input control (chromatin before immunoprecipitation)

  • Include IgG negative control

  • Consider using cells treated with CARM1 inhibitors as biological negative controls

  • Include a positive control such as a known H3R17me2a-enriched locus (e.g., estrogen-responsive genes after estrogen treatment)

• Validation Strategy: After ChIP, validate enrichment by qPCR at known target sites before proceeding to genome-wide analyses.

• Data Analysis: For ChIP-seq, use appropriate bioinformatic pipelines to identify H3R17me2a-enriched regions and correlate with gene expression data.

Research has shown that H3R17 methylation is enriched at the promoters of actively transcribed genes, particularly following hormone stimulation, and can be effectively detected using the Ab-17 antibody in ChIP experiments .

How can I distinguish between symmetric and asymmetric dimethylation of H3R17?

Distinguishing between symmetric and asymmetric dimethylation of H3R17 is important for understanding the specific biological roles of each modification. The HIST1H3A (Ab-17) antibody specifically recognizes the asymmetrically dimethylated form (H3R17me2a) and not the symmetrically dimethylated form (H3R17me2s) . To ensure proper distinction:

• Antibody Validation: Perform dot blot analysis using peptides with different methylation states, including unmethylated, monomethylated, asymmetrically dimethylated, and symmetrically dimethylated H3R17 peptides .

• Methyltransferase Specificity: CARM1 generates asymmetric dimethylation on H3R17, while other enzymes (like PRMT5) typically generate symmetric dimethylation on different residues. Using methyltransferase-specific inhibitors or knockdowns can help distinguish between these forms .

• Mass Spectrometry: For unambiguous identification, use mass spectrometry to distinguish between symmetric and asymmetric dimethylation based on their distinct fragmentation patterns.

• Functional Studies: Combine antibody-based detection with functional studies that link specific arginine methyltransferases to observed phenotypes.

A dot blot validation reported in the literature demonstrated that the Ab-17 antibody specifically recognized the asymmetrically dimethylated R17 peptide (lane 3) but not the unmodified peptide (lane 1), monomethylated R17 peptide (lane 2), or symmetrically dimethylated R17 peptide (lane 4) .

What experimental challenges might I encounter when detecting arginine methylation on histones in vivo?

Detecting arginine methylation on histones in vivo presents several challenges that researchers should be aware of:

• Low Abundance: Arginine methylation occurs at relatively low abundance compared to other histone modifications, making detection challenging. Only recently has methylation of arginine residues been detectable by sequencing of bulk purified histones .

• Antibody Specificity: Ensuring antibody specificity is critical as there can be cross-reactivity between different methylation states (mono-, di-, symmetric vs. asymmetric) and between different arginine residues within the same histone .

• Dynamic Nature: Arginine methylation is dynamically regulated, potentially with rapid turnover rates, making timing of experiments crucial.

• Extraction Methods: Standard histone extraction protocols may not efficiently preserve all arginine methylation marks, necessitating optimization of extraction methods.

• Cell-Type Specificity: The prevalence of H3R17 methylation can vary across cell types and physiological conditions, requiring careful selection of experimental systems .

• Detection Limitations: Traditional amino acid sequencing methods have limitations in detecting arginine methylation, which explains why this modification was not readily identified until specific antibodies became available .

To overcome these challenges, researchers have successfully used antibody-based approaches (like the Ab-17 antibody) that specifically recognize methylated R17, which has proven more sensitive than bulk histone sequencing for detecting this modification in vivo .

How does H3R17 methylation interact with other histone modifications in the context of gene regulation?

H3R17 methylation exists within a complex network of histone modifications that collectively regulate gene expression. Understanding these interactions is crucial for deciphering the histone code:

• Sequential Modification Patterns: Research suggests that H3R17 methylation by CARM1 may be influenced by prior acetylation events. The cooperative action of histone acetyltransferases and CARM1 has been observed in transcriptional activation contexts .

• Modification Cross-Talk: Evidence indicates possible synergy between H3R17 methylation and modifications like H3K18 acetylation during gene activation .

• Temporal Dynamics: Studies of hormone-responsive genes show that methylation at H3R17 coincides with gene activation, suggesting a temporal relationship with other activation-associated modifications .

• Competitive Modifications: Some modifications may compete for the same or nearby residues, creating mutual exclusivity that drives specific transcriptional outcomes.

• Reader Protein Recruitment: Different combinations of histone modifications, including H3R17 methylation, likely create binding surfaces for specific reader proteins that influence transcriptional outcomes.

To study these interactions, researchers can employ the following methodologies:

• Sequential ChIP (Re-ChIP): To determine co-occurrence of H3R17me2a with other modifications on the same nucleosomes

• Mass Spectrometry: To identify combinations of modifications on the same histone tail

• Functional Genomics: Combining ChIP-seq for multiple modifications with transcriptomics to correlate modification patterns with gene expression

These approaches, utilizing antibodies like the HIST1H3A (Ab-17) antibody, can help elucidate the complex interplay between H3R17 methylation and other histone modifications in gene regulation .

What are the optimal sample preparation methods for immunofluorescence using this antibody?

For optimal immunofluorescence results with the HIST1H3A (Ab-17) antibody, the following sample preparation protocol is recommended based on published methods:

• Fixation: Fix cells with 100% methanol for 5 minutes .

• Blocking: Incubate in 1% BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1 hour to permeabilize cells and block non-specific protein-protein interactions .

• Primary Antibody: Apply the antibody at 0.1 μg/ml concentration and incubate overnight at +4°C .

• Secondary Antibody: Use an appropriate secondary antibody such as Goat Anti-Rabbit IgG H&L (DyLight® 488) preadsorbed at 1/250 dilution for 1 hour .

• Counterstaining:

  • For membrane visualization: Alexa Fluor® 594 WGA at 1/200 dilution for 1 hour

  • For nuclear staining: DAPI at a concentration of 1.43μM

This protocol has been successfully used to visualize nuclear localization of H3R17me2a in MCF7 cells, with the methylation mark appearing primarily in the nuclear compartment .

How should I select positive and negative controls for experiments using this antibody?

Proper control selection is essential for interpreting results with the HIST1H3A (Ab-17) antibody:

Positive Controls:

• Cell Lines/Tissues: Use cell lines or tissues known to express active CARM1 and exhibit H3R17 methylation, such as:

  • MCF7 cells (especially when treated with estrogen)

  • HeLa cells

  • Human tonsil tissue sections

• Induced Systems: Hormone-treated cells where transcriptional activation is expected (e.g., estrogen-treated MCF7 cells for estrogen-responsive genes) .

• In vitro Methylated Substrates: Recombinant histone H3 methylated by CARM1 in vitro .

Negative Controls:

• Antibody Controls:

  • Peptide competition with the methylated R17 peptide to confirm specificity

  • IgG isotype control for non-specific binding

• Biological Controls:

  • Cell lines with CARM1 knockdown or knockout

  • Cell lines treated with CARM1 inhibitors

  • Recombinant unmethylated histone H3

• Peptide Controls for Validation:

  • Unmethylated H3 peptide

  • Monomethylated R17 peptide

  • Symmetrically dimethylated R17 peptide

  • Peptides with methylation at other residues

Published data shows that the antibody specifically recognizes asymmetrically dimethylated R17 on histone H3 and does not cross-react with unmethylated H3, symmetrically dimethylated R17, or methylation at other arginine residues .

What troubleshooting approaches can I use for inconsistent results with this antibody?

When encountering inconsistent results with the HIST1H3A (Ab-17) antibody, consider the following troubleshooting approaches:

• Antibody Storage and Handling:

  • Ensure proper storage at recommended temperature

  • Avoid repeated freeze-thaw cycles

  • Check expiration date and lot variability

• Sample Preparation Issues:

  • Histone extraction methods may impact modification preservation

  • Ensure complete protein denaturation for Western blots

  • Optimize fixation conditions for immunofluorescence/immunohistochemistry

• Signal Detection Problems:

  • For weak signals: Increase antibody concentration, extend incubation time, or use more sensitive detection systems

  • For high background: Increase blocking time, optimize washing steps, or decrease antibody concentration

  • For multiple bands: Verify specificity with peptide competition assays

• Biological Variables:

  • Cell density and growth conditions affect histone modification levels

  • Cell cycle phase impacts histone modification patterns

  • Verify CARM1 expression/activity in your experimental system

• Technical Considerations:

  • For Western blot: The predicted band size is 15 kDa, but observed bands may appear at 17 kDa, 55 kDa, 60 kDa, and 90 kDa

  • For ChIP: Optimize chromatin fragmentation and antibody concentration

  • For immunofluorescence: Ensure proper cell permeabilization

• Validation Approaches:

  • Perform dot blot against peptides with different methylation states

  • Use multiple antibodies targeting the same modification if available

  • Consider orthogonal detection methods (e.g., mass spectrometry)

Documentation of observed experimental variations can help identify patterns and resolve inconsistencies in results.

How can the HIST1H3A (Ab-17) antibody be used to study the role of CARM1 in gene regulation?

The HIST1H3A (Ab-17) antibody provides a powerful tool for investigating CARM1's role in gene regulation through the following approaches:

• ChIP-seq Analysis:

  • Map genome-wide distribution of H3R17me2a to identify CARM1 target genes

  • Compare H3R17me2a profiles before and after CARM1 knockdown/inhibition

  • Correlate H3R17me2a enrichment with transcriptional activity data

• Mechanistic Studies:

  • Use ChIP to examine the recruitment kinetics of CARM1 and the appearance of H3R17me2a during gene activation

  • Perform sequential ChIP to determine co-occupancy of CARM1, H3R17me2a, and transcription factors

• Signaling Pathway Analysis:

  • Examine changes in H3R17me2a in response to specific signaling events (e.g., hormone stimulation)

  • Compare H3R17me2a patterns in wild-type cells versus cells with mutations in upstream signaling components

• Protein Interaction Studies:

  • Identify proteins that interact with methylated H3R17 (reader proteins)

  • Investigate how H3R17 methylation affects the binding of other chromatin-associated proteins

• Functional Validation:

  • Use the antibody to validate CARM1 activity in various experimental models

  • Monitor H3R17me2a levels in response to CARM1 modulators

Research using this antibody has demonstrated that CARM1 is recruited to estrogen receptor-regulated promoters coincident with the appearance of H3R17 methylation, providing direct evidence for CARM1's role in transcriptional activation through histone modification .

What emerging applications exist for studying H3R17 methylation in disease contexts?

Emerging applications for studying H3R17 methylation in disease contexts include:

• Cancer Research:

  • Alterations in arginine methylation patterns have been implicated in various cancers

  • H3R17me2a levels may serve as potential biomarkers for hormone-responsive cancers

  • CARM1 overexpression has been observed in several cancer types, suggesting dysregulated H3R17 methylation

• Developmental Disorders:

  • Investigating the role of H3R17 methylation in developmental gene regulation

  • Examining potential aberrations in arginine methylation in developmental disorders

• Inflammatory Diseases:

  • Exploring connections between H3R17 methylation and inflammatory gene expression

  • Studying the impact of environmental factors on arginine methylation patterns

• Neurodegenerative Diseases:

  • Examining the role of histone arginine methylation in neurodegenerative processes

  • Investigating CARM1 function in neuronal gene expression and maintenance

• Methodological Approaches:

  • Immunohistochemistry: Using the Ab-17 antibody on tissue microarrays to correlate H3R17me2a with disease progression

  • ChIP-seq combined with RNA-seq: Identifying disease-specific gene networks regulated by H3R17 methylation

  • Single-cell approaches: Examining cell-type specific variations in H3R17 methylation in heterogeneous disease tissues

These applications leverage the specificity of the Ab-17 antibody to provide insights into the role of H3R17 methylation in pathological processes, potentially identifying new therapeutic targets or diagnostic markers.

How can I integrate H3R17 methylation data with other epigenomic datasets?

Integrating H3R17 methylation data with other epigenomic datasets provides comprehensive insights into chromatin regulation. Here are methodological approaches for such integration:

• Multi-omics Data Collection:

  • Generate ChIP-seq data for H3R17me2a using the Ab-17 antibody

  • Collect complementary datasets: other histone modifications, transcription factor binding, chromatin accessibility, RNA-seq

• Computational Integration Strategies:

  • Co-occurrence Analysis: Identify genomic regions where H3R17me2a co-occurs with other modifications

  • Correlation Analysis: Calculate genome-wide correlation between H3R17me2a and other epigenetic marks

  • Chromatin State Modeling: Use tools like ChromHMM to define chromatin states that include H3R17me2a

  • Trajectory Analysis: Examine temporal changes in H3R17me2a during biological processes

• Functional Associations:

  • Correlate H3R17me2a patterns with gene expression data

  • Identify enhancer regions marked by H3R17me2a and connect to target genes

  • Analyze transcription factor binding sites enriched in H3R17me2a-marked regions

• Visualization Approaches:

  • Generate heatmaps showing H3R17me2a distribution alongside other epigenetic marks

  • Create browser tracks for integrated visualization of multiple datasets

  • Develop composite profiles around features of interest (e.g., transcription start sites)

• Validation Experiments:

  • Confirm predicted interactions with sequential ChIP (Re-ChIP)

  • Validate functional relationships with CRISPR-based epigenome editing

  • Test predicted regulatory mechanisms with reporter assays

Research has shown that H3R17 methylation is associated with gene activation and may cooperate with other modifications like acetylation, suggesting a potential "cross-talk" between different histone modifications in transcriptional regulation .

What is the exact epitope recognized by the HIST1H3A (Ab-17) antibody?

The HIST1H3A (Ab-17) antibody recognizes a specific epitope on histone H3 where arginine 17 (R17) is asymmetrically dimethylated (H3R17me2a). The epitope corresponds to the N-terminal region of histone H3 surrounding R17. Based on the available information:

• Epitope Sequence: The antibody was raised against a synthetic peptide corresponding to amino acids 11-24 of histone H3, with R17 asymmetrically dimethylated .

• Specificity: Extensive validation through peptide competition assays and dot blot analyses confirms that the antibody specifically recognizes the asymmetrically dimethylated form of R17 and does not cross-react with:

  • Unmethylated R17

  • Monomethylated R17

  • Symmetrically dimethylated R17

  • Methylation at other arginine residues

• Production Method: The antibody was generated by immunizing rabbits with the methylated peptide. The resulting antiserum was purified through sequential affinity chromatography using columns with unmethylated and methylated peptides to isolate antibodies specific for the methylated form .

• Validated Recognition: The antibody has been validated to specifically recognize asymmetrically dimethylated R17 in multiple experimental contexts, including Western blot, immunofluorescence, chromatin immunoprecipitation, and peptide arrays .

This high specificity makes the antibody a valuable tool for detecting and studying this particular histone modification in various research applications.

What are the storage and handling recommendations to maintain antibody performance?

To maintain optimal performance of the HIST1H3A (Ab-17) antibody, follow these storage and handling recommendations:

• Storage Temperature:

  • Store at -20°C for long-term storage

  • Avoid repeated freeze-thaw cycles by aliquoting upon receipt

• Working Solution Preparation:

  • Dilute only the amount needed for immediate use

  • Prepare working solutions in appropriate buffers (PBS with 0.1% BSA recommended)

  • Keep diluted antibody on ice during experiment setup

• Stability Considerations:

  • Check expiration date before use

  • Monitor for signs of degradation (precipitates, loss of activity)

  • Record lot numbers for experimental reproducibility

• Handling Precautions:

  • Avoid contamination with bacteria or fungi

  • Wear gloves when handling antibody solutions

  • Centrifuge vials briefly before opening to collect solution at the bottom

• Application-Specific Storage:

  • For Western blotting: Can be stored at 4°C for up to 2 weeks after dilution

  • For immunofluorescence: Prepare fresh dilutions for each experiment

  • For ChIP applications: Prepare fresh dilutions on the day of the experiment