Formyl-HIST1H3A (K18) Antibody

What are the key modifications at the K18 position of Histone H3 and how do they affect chromatin function?

Histone H3 at lysine 18 (K18) can undergo several post-translational modifications, including mono-methylation, acetylation, and 2-hydroxyisobutyrylation. These modifications play central roles in transcription regulation, DNA repair, DNA replication, and chromosomal stability . Specifically, nucleosomes wrap and compact DNA into chromatin, limiting DNA accessibility to cellular machineries. The various modifications at K18 contribute to the "histone code" that regulates this DNA accessibility . For instance, acetylation at K18 is often associated with active transcription, while methylation may have different effects depending on the cellular context and the presence of other modifications.

How do researchers distinguish between different H3K18 modification-specific antibodies?

Researchers must carefully select antibodies that specifically recognize the exact modification of interest. The available antibodies targeting H3K18 modifications include:

• Anti-Histone H3 (mono methyl K18) antibodies, such as the rabbit recombinant monoclonal antibody [EPR17710]

• Anti-Histone H3 (acetyl K18) antibodies, like the rabbit recombinant monoclonal [EPR16595]

• Anti-HIST1H3A (2-hydroxyisobutyryl-K18) antibodies, including rabbit polyclonal options

When selecting an antibody, researchers should verify the specific epitope recognition through validation data provided by manufacturers, which often includes peptide array experiments demonstrating specificity for the modification of interest .

What are the validated applications for H3K18 modification antibodies?

Based on the search results, H3K18 modification antibodies have been validated for multiple applications:

The specific applications validated for each antibody should guide experimental design decisions .

What are the optimal protocols for Western blot experiments using H3K18 modification antibodies?

For Western blot analysis of H3K18 modifications, researchers should follow these key methodological steps based on validated protocols:

• Sample preparation: Use 30 μg of protein sample under reducing conditions

• Gel electrophoresis: Run on 5-20% SDS-PAGE gel at 70V (stacking gel) / 90V (resolving gel) 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/TBS for 1.5 hour at room temperature

• Primary antibody: Incubate with anti-H3K18 modification antibody (typically at 1:500 dilution) overnight at 4°C

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

• Secondary antibody: Probe with goat anti-rabbit IgG-HRP at 1:5000 dilution for 1.5 hour at room temperature

• Detection: Develop signal using enhanced chemiluminescent detection kit

The expected band size for H3K18-modified histone is approximately 15-17 kDa .

How should immunohistochemistry protocols be optimized for detecting H3K18 modifications in tissue sections?

For optimal IHC results with H3K18 modification antibodies:

• Section preparation: Use paraffin-embedded tissue sections

• Antigen retrieval: Perform heat-mediated antigen retrieval in EDTA buffer (pH 8.0, epitope retrieval solution)

• Blocking: Block the tissue section with 10% goat serum

• Primary antibody incubation: Incubate with anti-H3K18 modification antibody (typically at 1:500 dilution) overnight at 4°C

• Secondary antibody: Use peroxidase-conjugated goat anti-rabbit IgG and incubate for 30 minutes at 37°C

• Development: Develop using HRP-conjugated detection system with DAB as the chromogen

This protocol has been validated for brain tissue from both mouse and rat samples .

How can researchers verify the specificity of H3K18 modification antibodies?

Antibody specificity is critical for accurate interpretation of results. Researchers should:

• Perform peptide competition assays using modified and unmodified peptides

• Test antibodies on samples with known modification status as positive and negative controls

• Validate using multiple detection methods (e.g., both Western blot and immunofluorescence)

• Check for cross-reactivity with similar modifications at other lysine residues

• Use knockout/knockdown models where the modification enzyme is depleted

Manufacturers often provide cross-reactivity data showing specificity for the target modification versus other similar histone modifications .

What technical challenges should researchers anticipate when using ChIP with H3K18 modification antibodies?

When performing Chromatin Immunoprecipitation (ChIP) with H3K18 modification antibodies, researchers should consider:

• Fixation conditions affecting epitope accessibility

• Appropriate sonication parameters for optimal chromatin fragmentation

• Antibody concentration optimization to maximize specific binding while minimizing background

• Inclusion of appropriate controls, including input samples and IgG controls

• Sensitivity to different chromatin preparation methods

If combining with sequencing (ChIP-seq), additional considerations include library preparation quality and sequencing depth appropriate for the expected genomic distribution of the modification .

How do different H3K18 modifications interact with each other in the epigenetic code?

H3K18 can undergo various modifications that may interact functionally:

• Competition for the same lysine residue: Since K18 can only carry one modification at a time (methylation, acetylation, or hydroxyisobutyrylation), these modifications are mutually exclusive at any given histone molecule

• Sequential modifications: One modification may be replaced by another during biological processes like transcriptional activation

• Co-occurrence patterns: Certain H3K18 modifications may frequently co-occur with specific modifications at other histone residues

• Reading mechanisms: Different nuclear proteins ("readers") specifically recognize each type of modification

Understanding these interactions requires analyzing multiple modifications simultaneously, often using multiplexed antibody approaches or mass spectrometry .

What are the most common causes of false positives or inconsistent results when using H3K18 modification antibodies?

Common issues and their solutions include:

Regular quality control through positive and negative controls is essential for maintaining reproducible results .

How should researchers store and handle H3K18 modification antibodies to maintain optimal activity?

For optimal antibody performance:

• Store concentrated antibodies at -20°C for long-term storage (up to one year)

• For frequent use and short-term storage, store at 4°C for up to one month

• Avoid repeated freeze-thaw cycles, which can degrade antibody quality

• Some antibodies are supplied in 50% glycerol with preservatives like 0.03% Proclin 300 in PBS (pH 7.4)

• Always centrifuge briefly before opening vials to collect liquid at the bottom

Following these storage recommendations ensures maintained antibody activity and experimental reproducibility .

How are H3K18 modification antibodies being used in single-cell epigenomic analyses?

Recent advances in single-cell technologies have expanded the application of H3K18 modification antibodies:

• Single-cell CUT&Tag/CUT&RUN approaches allow profiling of histone modifications in individual cells

• Mass cytometry (CyTOF) with metal-conjugated antibodies enables quantification of multiple histone modifications simultaneously at the single-cell level

• Droplet-based microfluidic approaches combined with barcoding strategies permit high-throughput single-cell epigenomic profiling

These techniques require highly specific antibodies and often involve optimized fixation and permeabilization protocols to maintain cellular integrity while allowing antibody access .

What considerations are important when selecting controls for experiments using H3K18 modification antibodies?

Proper experimental controls are essential for accurate interpretation:

• Negative controls: Include isotype control antibodies to assess non-specific binding

• Positive controls: Use cell lines or tissues with known modification patterns

• Competitive inhibition: Perform peptide competition assays with modified and unmodified peptides

• Technical replicates: Include multiple technical replicates to assess experimental variability

• Biological replicates: Use samples from different biological sources to ensure reproducibility

• Cross-validation: Confirm findings using alternative detection methods when possible

Implementing comprehensive controls increases confidence in experimental results and facilitates troubleshooting when unexpected results occur .