Acetyl-HIST1H3A (K23) Antibody

What is Acetyl-HIST1H3A (K23) Antibody and what does it detect?

Acetyl-HIST1H3A (K23) antibody, also known as Acetyl-Histone H3-K23 antibody, is a research tool specifically designed to detect acetylation at lysine 23 on histone H3.1 protein. This antibody recognizes the post-translational modification where an acetyl group has been added to the lysine residue at position 23 of the histone H3 protein. Histone H3 is a core component of nucleosomes, which wrap and compact DNA into chromatin, thereby playing a central role in regulating DNA accessibility . The antibody is generated using synthetic acetylated peptides around the K23 position of human Histone H3 as immunogens, ensuring specificity for this particular modification .

What are the available formats of Acetyl-HIST1H3A (K23) antibodies?

Acetyl-HIST1H3A (K23) antibodies are available in both polyclonal and monoclonal formats, each with distinct characteristics:

Both formats are typically unconjugated (without fluorescent or enzymatic labels) and require secondary antibodies for detection, though conjugation services may be available for some products . The observed molecular weight of the detected protein is approximately 17kDa, while the calculated molecular weight is around 15-16kDa .

What biological processes involve H3K23 acetylation?

H3K23 acetylation is involved in several critical biological processes related to chromatin structure and function:

• Transcriptional regulation - H3K23ac contributes to creating open chromatin structures that facilitate gene expression

• DNA repair mechanisms - This modification has been linked to DNA damage response pathways

• Cell cycle regulation - Changes in H3K23 acetylation patterns occur during different phases of the cell cycle

• Developmental processes - Specific patterns of H3K23 acetylation are associated with cellular differentiation and development

Research indicates that H3K23 acetylation often occurs in conjunction with other histone modifications to form complex regulatory patterns that influence chromatin accessibility and gene expression . The presence of H3K23 acetylation is generally associated with transcriptionally active regions of the genome.

What are the recommended applications and dilutions for Acetyl-HIST1H3A (K23) antibodies?

Acetyl-HIST1H3A (K23) antibodies can be used in multiple experimental approaches, each requiring specific dilutions for optimal results:

The appropriate dilution should be determined empirically for each application and specific antibody, as sensitivity can vary between different antibody clones and lots .

How should samples be prepared for optimal detection of H3K23 acetylation?

Proper sample preparation is critical for accurate detection of H3K23 acetylation:

• Cell/Tissue Lysis: Use specialized histone extraction buffers containing histone deacetylase inhibitors (e.g., sodium butyrate, trichostatin A) to preserve acetylation marks.

• Treatment Conditions: For positive controls or to enhance acetylation signals, cells can be treated with histone deacetylase inhibitors such as trichostatin A (TSA). For example, HeLa, NIH/3T3, and C6 cells treated with 1 μM TSA for 18 hours show enhanced H3K23 acetylation signals .

• Protein Quantification: Accurate protein quantification is essential, with recommended loading of 25μg protein per lane for Western blots to ensure consistent results .

• Blocking Conditions: For Western blots, 3% nonfat dry milk in TBST has been shown to provide adequate blocking while maintaining specific signal detection .

• Fixation for Microscopy: For immunofluorescence applications, appropriate fixation (typically 4% paraformaldehyde) and permeabilization protocols must be followed to maintain nuclear architecture while allowing antibody access to chromatin.

What controls should be included when using Acetyl-HIST1H3A (K23) antibody?

Including appropriate controls is essential for interpreting results with Acetyl-HIST1H3A (K23) antibody:

• Positive Controls:

  • Cell lines known to exhibit H3K23 acetylation (HeLa, NIH/3T3, C6)

  • Cells treated with histone deacetylase inhibitors like TSA (1 μM, 18 hours)

  • Recombinant acetylated histone peptides

• Negative Controls:

  • Primary antibody omission

  • Isotype control (rabbit IgG)

  • Cells treated with histone acetyltransferase inhibitors

  • Competitive blocking with acetylated peptides

• Specificity Controls:

  • Peptide competition assays using both H3K23ac and unrelated acetylated peptides

  • Western blot comparison with other histone H3 antibodies

  • Dot-blot analysis against multiple modified histone peptides to confirm specificity

How does cross-reactivity affect Acetyl-HIST1H3A (K23) antibody performance?

Cross-reactivity is a significant concern with histone modification antibodies, including those targeting H3K23ac:

• Neighboring Modifications: H3K23ac antibodies can be affected by modifications at neighboring residues. Similar to what has been observed with other histone antibodies, the presence of phosphorylation at adjacent serine residues might affect binding efficiency .

• Similar Acetylation Sites: Antibodies targeting H3K23ac may cross-react with other acetylated lysine residues on histone H3, particularly those with similar surrounding sequences (e.g., H3K18ac, H3K27ac).

• Iterative Acetylation Effects: As observed with H4 acetylation antibodies, H3 acetylation antibodies may show increased signal when multiple acetylation sites are present on the same histone tail. This "iterative acetylation effect" can result in stronger signals that don't necessarily reflect only the specific acetylation site being targeted .

• Off-target Recognition: Some histone antibodies have demonstrated unexpected cross-reactivity with unrelated modifications. For example, certain H3K27me3 antibodies have been shown to cross-react with H3K4me3 marks, especially when these marks appear in combination with neighboring acetylation modifications .

What techniques can be used to validate the specificity of Acetyl-HIST1H3A (K23) antibodies?

Multiple complementary approaches should be used to validate antibody specificity:

• Peptide Microarrays: Comprehensive testing using arrays containing various histone modifications can reveal potential cross-reactivity. These arrays allow for testing multiple modification states and combinatorial modifications simultaneously .

• Dot Blot Analysis: Testing reactivity against a panel of modified peptides can identify potential cross-reactivity. For example, dot-blot analysis can demonstrate whether an H3K23ac antibody binds only to H3K23ac peptides or also recognizes other modified residues .

• Genetic Controls: Using cells with mutations at specific lysine residues (K→R mutations) or cells deficient in specific histone acetyltransferases can provide genetic validation of antibody specificity.

• Mass Spectrometry Correlation: Comparing antibody-based detection with mass spectrometry quantification of histone modifications can provide independent validation of antibody specificity and accuracy.

• Western Blot with Competing Peptides: Pre-incubating the antibody with increasing concentrations of specific and non-specific peptides can demonstrate binding specificity through signal reduction.

How does H3K23 acetylation interact with other histone modifications?

H3K23 acetylation functions within a complex network of histone modifications:

• Co-occurrence Patterns: H3K23ac often co-occurs with other active marks such as H3K9ac, H3K14ac, and H3K4me3, collectively forming transcriptionally permissive chromatin environments.

• Modification Crosstalk: The presence of specific modifications can influence the deposition or removal of H3K23ac. For example, phosphorylation of neighboring residues may affect the ability of acetyltransferases to modify H3K23.

• Sequential Modifications: In some contexts, H3K23ac may be part of a sequential modification pattern, where one modification leads to the recruitment of enzymes that deposit subsequent modifications.

• Binary Switches: H3K23 can also be methylated, creating a potential binary switch where acetylation and methylation are mutually exclusive and potentially drive different biological outcomes.

• Specific Binding Proteins: Different protein complexes specifically recognize H3K23ac within particular modification contexts, leading to context-specific downstream effects.

Understanding these interactions is critical for interpreting experimental results and building comprehensive models of chromatin regulation.

What are common issues when using Acetyl-HIST1H3A (K23) antibody in Western blots?

Several common issues can affect Western blot results with Acetyl-HIST1H3A (K23) antibody:

• Weak or No Signal:

  • Insufficient histone extraction

  • Degradation of acetylation marks during sample preparation (add HDAC inhibitors)

  • Antibody concentration too low (try 1:500 instead of 1:1000)

  • Incomplete transfer of histones to membrane (optimize transfer conditions for low molecular weight proteins)

• High Background:

  • Insufficient blocking (increase blocking time or concentration)

  • Antibody concentration too high (increase dilution)

  • Excessive exposure time

  • Non-specific binding (try different blocking agents, add 0.05% BSA to antibody dilution)

• Multiple Bands:

  • Cross-reactivity with other acetylated histones

  • Histone degradation products

  • Post-translational modification heterogeneity

• Inconsistent Results:

  • Batch-to-batch variation in antibodies

  • Inconsistent sample preparation

  • Variable transfer efficiency

  • Changes in histone acetylation during sample handling

For optimal results, researchers should use freshly prepared samples with HDAC inhibitors, optimize antibody concentration for each new lot, and include appropriate positive controls .

Why might ChIP experiments with Acetyl-HIST1H3A (K23) antibody fail?

ChIP experiments with Acetyl-HIST1H3A (K23) antibody can fail for several reasons:

• Chromatin Preparation Issues:

  • Inadequate crosslinking (optimize formaldehyde concentration and time)

  • Improper chromatin fragmentation (aim for 200-500bp fragments)

  • Loss of acetylation marks during chromatin preparation

• Immunoprecipitation Problems:

  • Insufficient antibody amount (use 2μg antibody per 5-10μg of chromatin)

  • Poor antibody binding efficiency

  • Inadequate washing conditions (too stringent or too gentle)

  • Non-specific binding to beads (pre-clear chromatin and use appropriate blocking)

• PCR Detection Challenges:

  • Inefficient DNA purification after ChIP

  • Inappropriate primer design

  • Inhibitory contaminants in eluted DNA

• Biological Limitations:

  • Low abundance of H3K23ac at the genomic regions of interest

  • Cell type-specific patterns of H3K23ac

  • Dynamic changes in H3K23ac during cell cycle or experimental treatment

How can neighboring modifications affect Acetyl-HIST1H3A (K23) antibody binding?

The binding efficiency and specificity of Acetyl-HIST1H3A (K23) antibodies can be significantly affected by neighboring modifications:

• Adjacent Phosphorylation: Similar to observations with other histone antibodies, phosphorylation at nearby residues (such as S22 or T24) may interfere with antibody binding. For example, some H3K9me3 antibodies are insensitive to neighboring H3S10 phosphorylation while others are strongly affected .

• Neighboring Acetylation: Additional acetylation marks near K23 might enhance antibody binding through cooperative effects, similar to what has been observed with H4 acetylation antibodies that show enhanced signal with increasing acetylation content .

• Neighboring Methylation: Methylation at nearby lysine residues might alter the epitope structure, affecting antibody recognition of H3K23ac.

• Combinatorial Effects: The combination of various modifications around K23 can create complex epitope structures that may enhance or reduce antibody binding in unpredictable ways.

Researchers should be aware of these potential effects and validate their antibodies using peptide arrays with combinatorial modifications to understand how neighboring modifications affect specific antibody binding .

How can Acetyl-HIST1H3A (K23) antibody be used in disease research?

Acetyl-HIST1H3A (K23) antibodies can provide valuable insights in various disease contexts:

• Cancer Research:

  • Mapping changes in H3K23ac profiles between normal and cancer cells

  • Correlating H3K23ac patterns with oncogene activation or tumor suppressor silencing

  • Monitoring epigenetic alterations during cancer progression and in response to therapy

  • Identifying potential biomarkers based on H3K23ac distribution

• Neurodegenerative Disorders:

  • Examining H3K23ac changes in models of Alzheimer's, Parkinson's, and other neurodegenerative diseases

  • Investigating the relationship between histone acetylation and neuronal gene expression patterns

  • Evaluating the effects of HDAC inhibitors as potential therapeutic agents

• Inflammatory and Autoimmune Diseases:

  • Studying H3K23ac dynamics during immune cell activation and differentiation

  • Mapping acetylation changes associated with aberrant inflammatory responses

  • Identifying epigenetic signatures of chronic inflammation

• Developmental Disorders:

  • Characterizing H3K23ac patterns during normal and abnormal development

  • Investigating the impact of environmental factors on histone acetylation profiles

  • Studying the role of H3K23ac in cellular differentiation and tissue-specific gene expression

These applications require careful experimental design and often benefit from integrating multiple approaches such as ChIP-seq, RNA-seq, and proteomics to build comprehensive understanding of the role of H3K23ac in disease processes .

What are cutting-edge techniques that utilize Acetyl-HIST1H3A (K23) antibody?

Several advanced techniques leverage Acetyl-HIST1H3A (K23) antibodies for sophisticated epigenetic analysis:

• CUT&RUN (Cleavage Under Targets and Release Using Nuclease):

  • Offers higher signal-to-noise ratio than traditional ChIP

  • Requires fewer cells and less antibody

  • Provides higher resolution mapping of H3K23ac genomic distribution

• CUT&Tag (Cleavage Under Targets and Tagmentation):

  • Combines antibody binding with tagmentation for streamlined workflow

  • Enables single-cell epigenomic profiling of H3K23ac

  • Further improves sensitivity compared to CUT&RUN

• ChIC/CUT-ChIC (Chromatin Immunocleavage):

  • Uses protein A-MNase fusion proteins for targeted chromatin cleavage

  • Provides ultra-high resolution mapping of H3K23ac sites

• ChIP-SICAP (Selective Isolation of Chromatin-Associated Proteins):

  • Identifies proteins that interact with H3K23ac-marked chromatin regions

  • Helps elucidate the protein complexes recruited by H3K23ac

• Advanced Imaging Techniques:

  • Super-resolution microscopy for visualizing H3K23ac distribution in nuclear architecture

  • Live-cell imaging using engineered antibody fragments to track H3K23ac dynamics

• Single-Cell Approaches:

  • scChIP-seq for mapping H3K23ac in individual cells

  • Spatial epigenomics techniques to preserve tissue context while mapping H3K23ac

These emerging techniques continue to expand our understanding of the dynamic role of H3K23 acetylation in chromatin regulation and gene expression.