Acetyl-HIST1H3A (K122) Antibody

What is Acetyl-HIST1H3A (K122) and what role does it play in chromatin regulation?

Acetyl-HIST1H3A (K122) refers to the acetylation of lysine 122 on histone H3, a core component of nucleosomes. Nucleosomes wrap and compact DNA into chromatin, which limits DNA accessibility to cellular machineries that require DNA as a template. This specific acetylation plays a central role in transcription regulation, DNA repair, DNA replication, and chromosomal stability .

The acetylation at K122 is part of the complex set of post-translational modifications of histones, often referred to as the "histone code," which regulates DNA accessibility through both direct structural effects and recruitment of chromatin remodeling factors . This specific modification is particularly important for chromatin structure and gene expression regulation.

What applications are suitable for Acetyl-HIST1H3A (K122) antibodies?

Acetyl-HIST1H3A (K122) antibodies have been validated for multiple research applications:

These applications enable researchers to study the presence, abundance, and localization of H3K122ac in various experimental contexts .

How does species reactivity affect experimental design with Acetyl-HIST1H3A (K122) antibodies?

Different Acetyl-HIST1H3A (K122) antibodies exhibit varying species reactivity profiles that must be considered when designing experiments:

When working with non-human models, it's essential to verify antibody reactivity or choose an antibody with confirmed cross-reactivity to your species of interest. The high conservation of histone H3 sequences across species often allows for cross-reactivity, but validation is recommended for each new experimental system .

What are the optimal sample preparation protocols for detecting Acetyl-HIST1H3A (K122) in various applications?

For successful detection of Acetyl-HIST1H3A (K122), sample preparation is critical:

For Western Blotting:

• Extract histones using acid extraction methods (0.2N HCl or 0.4N H₂SO₄) to efficiently isolate histones from chromatin

• Alternatively, prepare whole cell lysates in buffers containing histone deacetylase inhibitors (e.g., sodium butyrate, trichostatin A)

• Use SDS-PAGE with 15-18% gels to achieve optimal separation of the low molecular weight (15-17 kDa) histone proteins

• Transfer to PVDF membranes (preferred over nitrocellulose for small proteins)

• Block with 5% BSA rather than milk (milk contains proteins that may have biotin and cause background)

For Immunohistochemistry:

• Fix tissues with formalin and embed in paraffin

• Perform antigen retrieval (typically heat-mediated in citrate buffer pH 6.0) to expose epitopes

• Include a permeabilization step (0.2-0.5% Triton X-100) to ensure nuclear access

• Block with appropriate sera to reduce background

Maintaining acetylation status during sample preparation is crucial, so samples should be processed quickly and kept cold, with histone deacetylase inhibitors included in all buffers .

How can researchers validate the specificity of Acetyl-HIST1H3A (K122) antibodies?

Validating antibody specificity is essential for reliable research results:

• Peptide Competition Assay:

  • Pre-incubate the antibody with excess acetylated K122 peptide

  • Compare results with and without peptide competition

  • Signal should be significantly reduced in the presence of competing peptide

• Positive and Negative Controls:

  • Use samples with known high levels of H3K122ac (e.g., samples treated with histone deacetylase inhibitors)

  • Compare with samples where K122 acetylation is reduced (e.g., using histone acetyltransferase inhibitors)

• Multiple Antibody Validation:

  • Compare results using antibodies from different sources targeting the same modification

  • Consistent results across different antibodies increase confidence in specificity

• Knockout/Knockdown Controls:

  • Where possible, use genetic models where key acetyltransferases responsible for K122 acetylation are depleted

These validation steps help ensure that the observed signals are truly representative of H3K122 acetylation rather than non-specific binding .

How does Acetyl-HIST1H3A (K122) function in the context of the histone code?

Acetyl-HIST1H3A (K122) functions within the broader context of the histone code, an intricate system of post-translational modifications that collectively regulate chromatin structure and function:

• Location Significance: K122 is positioned at the nucleosome dyad axis, making its acetylation particularly important for nucleosome stability and DNA unwrapping

• Cross-talk with Other Modifications:

  • H3K122ac often co-occurs with other active chromatin marks such as H3K27ac and H3K4me3

  • The presence of H3K122ac may influence how other modifications are recognized by reader proteins

• Functional Consequences:

  • Weakens histone-DNA interactions directly at the dyad axis

  • Facilitates transcription factor binding to otherwise inaccessible DNA regions

  • Contributes to nucleosome disassembly during processes requiring DNA access

• Regulatory Enzymes:

  • Written by histone acetyltransferases including p300/CBP

  • Removed by specific histone deacetylases

  • Recognized by bromodomain-containing proteins

Understanding H3K122ac in this integrated context is essential for interpreting its role in chromatin regulation and gene expression .

What are the considerations for using Acetyl-HIST1H3A (K122) antibodies in ChIP and ChIP-seq experiments?

Chromatin Immunoprecipitation (ChIP) with Acetyl-HIST1H3A (K122) antibodies requires careful optimization:

• Crosslinking Conditions:

  • Standard 1% formaldehyde for 10 minutes may be sufficient

  • For deeper analysis of nucleosome dynamics, dual crosslinking with EGS followed by formaldehyde may better preserve nucleosome structure

• Sonication Parameters:

  • Optimize to achieve 200-500 bp fragments

  • Over-sonication can disrupt nucleosome structure and epitope integrity

• Antibody Selection:

  • Choose antibodies specifically validated for ChIP applications

  • Polyclonal antibodies may provide better coverage of the epitope in crosslinked chromatin

• Controls:

  • Include input controls, IgG controls, and spike-in normalization

  • Use positive control regions known to be enriched for H3K122ac

• Data Analysis Considerations:

  • H3K122ac often presents as broad peaks rather than sharp signals

  • Integration with other histone marks helps contextual interpretation

  • Normalize to nucleosome occupancy data when available

For ChIP-seq specifically, antibodies should be validated for this application, as noted for several products in the Cell Signaling Technology Acetyl-Histone H3 Antibody Sampler Kit .

How does Acetyl-HIST1H3A (K122) pattern change during cell differentiation and disease progression?

The dynamic nature of H3K122ac during cellular processes provides important insights:

• Cell Differentiation:

  • H3K122ac undergoes significant redistribution during differentiation

  • Initially present at promoters of pluripotency genes in stem cells

  • Shifts to lineage-specific genes during differentiation

  • Often precedes other activating marks during gene activation events

• Disease States:

  • Cancer cells frequently show altered H3K122ac patterns

  • Immunohistochemical analysis in human breast cancer tissue shows distinct H3K122ac distribution patterns compared to normal tissue

  • Changes in global H3K122ac levels can reflect altered activity of specific histone acetyltransferases or deacetylases

• Monitoring Methods:

  • Western blotting for global changes in H3K122ac levels

  • ChIP-seq for genome-wide redistribution analysis

  • Immunohistochemistry for spatial distribution in tissue contexts

These dynamic changes make H3K122ac a valuable epigenetic marker for studying cellular transitions and disease mechanisms .

What are common problems when working with Acetyl-HIST1H3A (K122) antibodies and how can they be resolved?

Researchers frequently encounter these challenges when working with H3K122ac antibodies:

• Weak or No Signal in Western Blot:

  • Ensure proper histone extraction (acid extraction is often more effective than standard RIPA buffers)

  • Include HDAC inhibitors in all buffers to preserve acetylation

  • Try longer primary antibody incubation (overnight at 4°C)

  • Optimize antibody concentration (try 1:500 instead of 1:1000)

• High Background in Immunostaining:

  • Increase blocking time and concentration (5% BSA or normal serum)

  • Reduce primary antibody concentration

  • Include additional washing steps

  • Use more specific secondary antibodies

• Inconsistent ChIP Results:

  • Optimize crosslinking time specifically for H3K122ac

  • Ensure complete nuclear lysis

  • Pre-clear chromatin with protein A/G beads

  • Increase antibody amount or incubation time

• Variability Between Experiments:

  • Standardize cell culture conditions (density, passage number)

  • Use fresh antibody aliquots

  • Include positive control samples in each experiment

  • Maintain consistent sample processing times

Addressing these common issues methodically will improve the reliability and reproducibility of experiments using Acetyl-HIST1H3A (K122) antibodies .

How can researchers quantitatively analyze Acetyl-HIST1H3A (K122) levels across different experimental conditions?

Quantitative analysis of H3K122ac requires careful experimental design and appropriate normalization:

• Western Blot Quantification:

  • Normalize to total H3 loading (use parallel blots or strip and reprobe)

  • Use recombinant acetylated standards for absolute quantification

  • Employ fluorescent secondary antibodies for wider linear range of detection

  • Analyze using software like ImageJ with appropriate background correction

• Immunofluorescence Quantification:

  • Use identical microscopy settings across all samples

  • Quantify nuclear fluorescence intensity using software like CellProfiler

  • Normalize to DAPI or total H3 signal

  • Include multiple fields and biological replicates

• ChIP-qPCR Quantification:

  • Express as percent input or fold enrichment over IgG

  • Include multiple primer sets targeting both positive and negative regions

  • Use spike-in controls for cross-sample normalization

• Mass Spectrometry Approaches:

  • Most accurate for absolute quantification

  • Requires specialized equipment and expertise

  • Can simultaneously measure multiple histone modifications

What emerging technologies are advancing research on Acetyl-HIST1H3A (K122)?

Research on H3K122ac is being advanced by several cutting-edge technologies:

• CUT&RUN and CUT&Tag:

  • More sensitive alternatives to traditional ChIP

  • Require fewer cells and provide higher signal-to-noise ratio

  • Some antibodies from Cell Signaling Technology have been validated for these techniques

• Single-Cell Epigenomics:

  • Allows examination of H3K122ac heterogeneity within populations

  • Combines with transcriptomics for multi-omic analysis at single-cell level

• Live-Cell Imaging of Histone Modifications:

  • Uses engineered antibody fragments or modification-specific binding domains

  • Enables dynamic tracking of H3K122ac in living cells

• Long-Read Sequencing:

  • Improves the resolution of H3K122ac distribution

  • Allows correlation with other histone marks over longer genomic regions

These technological advances will continue to deepen our understanding of the dynamics and functional significance of H3K122 acetylation in diverse biological contexts .

What are the key considerations for integrating Acetyl-HIST1H3A (K122) data with other epigenetic marks?

Integrative analysis of H3K122ac with other epigenetic data provides comprehensive insights:

• Data Normalization Approaches:

  • Account for differences in antibody efficiencies

  • Normalize to appropriate controls (input, IgG, spike-ins)

  • Consider batch effects when combining datasets

• Correlation Analysis:

  • Determine co-occurrence patterns with other histone marks

  • Identify antagonistic relationships between modifications

  • Calculate correlation coefficients at different genomic features

• Functional Genomics Integration:

  • Combine with transcriptome data to link H3K122ac to gene expression

  • Integrate with chromatin accessibility data (ATAC-seq, DNase-seq)

  • Correlate with transcription factor binding profiles

• Visualization and Analysis Tools:

  • Use genome browsers with multiple track display capabilities

  • Employ clustering algorithms to identify patterns

  • Apply machine learning approaches for predictive modeling

This integrative approach positions H3K122ac research within the broader context of epigenetic regulation, revealing its unique contributions to chromatin function and gene expression control .