Propionyl-HIST1H4A (K5) Antibody

What is Propionyl-HIST1H4A (K5) Antibody and what modification does it detect?

Propionyl-HIST1H4A (K5) Antibody is a research reagent that specifically recognizes histone H4 propionylated at lysine 5. This polyclonal antibody, typically raised in rabbits, is generated using synthetic peptides containing propionylated K5 as immunogens. The antibody detects endogenous levels of this specific post-translational modification, which is a critical component of the histone code that regulates chromatin structure and function .

Histone H4 is a core component of nucleosomes that wrap and compact DNA into chromatin, limiting DNA accessibility to cellular machineries. This plays a central role in transcription regulation, DNA repair, DNA replication, and chromosomal stability. DNA accessibility is regulated through post-translational modifications of histones, including propionylation .

How does propionylation differ from other histone acylation modifications?

Propionylation involves the addition of a propionyl group (CH₃CH₂CO-) to lysine residues, distinguishing it from:

While all these modifications neutralize the positive charge of lysine residues, their distinct structures likely recruit different effector proteins and influence chromatin structure and function differently . Research suggests propionylation may function as a unique regulatory mechanism beyond acetylation due to its slightly more hydrophobic nature.

What are the key applications where Propionyl-HIST1H4A (K5) antibodies have been validated?

Based on multiple product validations, these antibodies have been confirmed effective in:

Researchers should optimize these dilutions for their specific experimental conditions .

How should I design controls for experiments using Propionyl-HIST1H4A (K5) antibody?

Implementing appropriate controls is crucial for reliable results:

Positive Controls:

• Cell lines treated with propionyl-CoA or sodium propionate to increase global propionylation levels

• Synthetic propionylated H4K5 peptides

Negative Controls:

• Unmodified histone H4 peptides

• Cells with enzymatic pathways that remove propionylation upregulated

• Peptide competition assays where the antibody is pre-incubated with propionylated peptides

Cross-Reactivity Controls:

• Peptides containing different histone modifications (acetylation, crotonylation) at the same position

• Peptides with propionylation at different lysine residues

Technical Controls:

• IgG control antibodies in immunoprecipitation experiments

• Input chromatin samples in ChIP assays

• Loading controls for Western blots (total H4 or other housekeeping proteins)

What optimization strategies improve ChIP experiments with Propionyl-HIST1H4A (K5) antibody?

For optimal ChIP results with histone modification antibodies:

• Crosslinking optimization:

  • Test different formaldehyde concentrations (0.5-1%)

  • Optimize crosslinking time (5-15 minutes) to prevent over-crosslinking

• Chromatin fragmentation:

  • Target 200-500 bp fragments

  • Verify fragmentation by agarose gel electrophoresis

  • Adjust sonication time, amplitude, and cycle number

• Antibody parameters:

  • Titrate antibody amount (2-10 μg per ChIP)

  • Include pre-clearing steps with protein A/G beads

  • Extend antibody incubation time (overnight at 4°C)

• Washing optimization:

  • Increase stringency gradually with buffers containing different salt concentrations

  • Test detergent concentrations to reduce background

• Enrichment analysis:

  • Include known positive regions (from published studies)

  • Normalize to input and IgG control

  • Calculate percent input for quantification

ChIP-seq studies have revealed that other H4 modifications (e.g., acetylation at K8 and K16) are enriched around transcription start sites, providing potential positive control regions for comparison .

How should I analyze and interpret Western blot results using this antibody?

For accurate Western blot analysis:

• Expected results:

  • Predicted molecular weight of histone H4: 11-12 kDa

  • Observed band size typically matches predicted (12 kDa)

  • May observe slight shifts due to additional modifications

• Quantification approach:

  • Always normalize to total H4 levels (using pan-H4 antibody)

  • Include loading controls (β-actin, GAPDH)

  • Use linear range of detection (avoid oversaturation)

  • Apply densiometric analysis with appropriate software

• Result interpretation:

  • Increased signal indicates higher propionylation levels

  • Compare experimental conditions to baseline

  • Consider biological significance of fold changes

  • Cross-validate with other techniques

Example Western blot data from search result #10 shows clear detection of propionylated H4K5 in various cell lines, including HeLa, 293, Jurkat, and HepG2 .

What are the key considerations for interpreting ChIP-seq data obtained with this antibody?

ChIP-seq analysis for histone modifications requires careful interpretation:

• Quality control metrics:

  • Fragment size distribution

  • Library complexity

  • Peak enrichment relative to input

  • Reproducibility between replicates

• Peak calling parameters:

  • Appropriate tools (MACS2, SICER for broad peaks)

  • FDR thresholds (typically 0.01-0.05)

  • Peak width considerations

• Genomic distribution analysis:

  • Enrichment at promoters, enhancers, gene bodies

  • Correlation with transcription start sites

  • Co-occurrence with other histone marks

  • Overlap with transcription factor binding sites

• Functional interpretation:

  • Correlation with gene expression data

  • Gene ontology/pathway analysis of marked genes

  • Cell-type specific patterns

  • Comparison with published datasets

Based on studies of related histone modifications, propionylation patterns may correlate with specific genomic features and regulatory functions .

How do propionylation levels compare across different cell types and physiological conditions?

Propionylation patterns show context-dependent variation:

Research indicates propionylation is dynamically regulated and may respond to metabolic states, as propionyl-CoA serves as the donor for this modification. Future studies should investigate how propionylation changes with:

• Cell cycle progression

• Differentiation status

• Nutrient availability

• Disease states

This remains an active area of investigation requiring further characterization .

What enzymes regulate histone propionylation, and how can they be experimentally manipulated?

Histone propionylation is regulated by writer and eraser enzymes:

Writers (Propionyltransferases):

• Several histone acetyltransferases (HATs) demonstrate dual activity

• p300/CBP exhibits significant propionyltransferase activity

• GCN5 and HAT1 show KPT activity almost equal to their KAT activity

• PCAF shows KPT activity at ~40% of its KAT activity

• MYST family members (MOF, HBO1, MOZ) show strong KPT activity with KPT/KAT ratios of 0.84, 0.99, and 1.04 respectively

Erasers (Depropionylases):

• Certain HDACs can remove propionyl groups

• Sirtuin family members may have depropionylase activity

Experimental manipulation:

• Overexpression systems for writer enzymes

• CRISPR-Cas9 knockout of enzymes

• Chemical inhibitors of HATs/HDACs

• Metabolic manipulation of propionyl-CoA levels

• In vitro enzyme assays to measure activity

Search result #15 notes: "The KPT activity of GCN5 and HAT1 is almost equally strong compared with their KAT activity, whereas the KPT activity of PCAF and p300 is about 40 and 30% of their acetyltransferase activity."

How does propionylation at H4K5 interact with other histone modifications in the histone code?

Histone modifications function as part of a complex, interdependent network:

• Modification cross-talk:

  • Physical occlusion: Propionylation prevents other modifications at the same residue

  • Enzymatic influence: Nearby modifications can affect enzyme binding and activity

  • Sequential modifications: One modification may predispose the region to subsequent modifications

• Neighboring residue effects:

  • H4K5 propionylation may influence modification of adjacent residues (K8, K12)

  • Search result #7 indicates that for acetylation: "an H4K5 acetylation-specific antibody CMA405 reacted with K5ac only when the neighboring K8 was unacetylated"

  • This suggests the importance of understanding combinatorial patterns

• Functional consequences:

  • Different combinations of modifications create unique binding surfaces

  • Reader proteins may recognize specific patterns rather than individual marks

  • Modifications work together to establish chromatin states

• Analytical challenges:

  • Need for specialized antibodies that recognize specific modification combinations

  • Mass spectrometry approaches to identify co-occurring modifications

  • Computational methods to decipher modification patterns

What are emerging techniques beyond antibody-based methods for studying histone propionylation?

Research is advancing through novel methodologies:

• Mass spectrometry innovations:

  • Data-independent acquisition (DIA) methods improve identification of modified peptides

  • Parallel reaction monitoring for targeted quantification

  • Top-down proteomics to analyze intact histones with all modifications

  • Example from search result #16: "An interactive mass spectrometry atlas of histone posttranslational modifications"

• Chemical biology approaches:

  • Clickable propionyl analogs for bioorthogonal labeling

  • Proximity labeling methods to identify proteins interacting with propionylated histones

  • Chemical probes for specific reader domains

• Structural biology techniques:

  • Cryo-EM studies of modified nucleosomes

  • X-ray crystallography of reader proteins bound to propionylated peptides

  • NMR analysis of modification-induced structural changes

• Single-molecule approaches:

  • FRET-based sensors for real-time monitoring

  • Single-molecule imaging of chromatin dynamics

  • Nanopore sequencing for direct detection of modifications

• Computational methods:

  • Machine learning algorithms to predict modification patterns

  • Network analysis of modification interactions

  • Integrated multi-omics data analysis

How do tissue-specific patterns of H4K5 propionylation correlate with gene expression programs?

Tissue-specific propionylation patterns represent a frontier in epigenetic research:

• Current understanding:

  • Limited data exists on tissue-specific H4K5 propionylation patterns

  • Patterns likely correlate with metabolic states specific to tissues

  • May regulate tissue-specific gene expression programs

• Experimental approaches:

  • ChIP-seq across tissue types to map modification landscapes

  • Integration with RNA-seq data to correlate with expression

  • Single-cell approaches to capture cellular heterogeneity

  • Developmental time course studies

• Functional hypotheses:

  • Tissue-specific propionylation may mark lineage-specific genes

  • Could represent a mechanism linking metabolism to gene regulation

  • May be involved in cellular memory during development

• Disease relevance:

  • Altered propionylation patterns may contribute to pathologies

  • Could represent biomarkers for disease states

  • Potential therapeutic target in epigenetic therapies

This area requires further investigation with comprehensive tissue analyses using both antibody-based and mass spectrometry approaches .

What are common causes for high background or weak signal when using Propionyl-HIST1H4A (K5) antibody?

Troubleshooting antibody-based experiments:

High Background Issues:

• Insufficient blocking - Extend blocking time or try alternative blocking agents

• Excessive antibody concentration - Perform titration experiments

• Inadequate washing - Increase wash times/volumes and add detergent

• Cross-reactivity - Validate specificity with peptide arrays

• Non-specific binding - Pre-clear samples with protein A/G beads

Weak Signal Problems:

• Low abundance of modification - Verify presence with MS or enrichment

• Epitope masking - Optimize fixation or antigen retrieval

• Antibody degradation - Check storage conditions and expiration

• Suboptimal incubation - Extend incubation time or adjust temperature

• Inefficient protein extraction - Modify extraction protocol for histones

Optimization Strategies:

• Test multiple antibody concentrations

• Adjust incubation times and temperatures

• Try different detection systems

• Include positive controls

• Verify target protein expression levels

How does sample preparation affect the detection of propionylated histones?

Sample preparation is critical for accurate detection:

• Histone extraction methods:

  • Acid extraction preserves modifications but may cause some losses

  • Triton extraction maintains nuclear integrity but may be less efficient

  • Commercial kits provide standardized approaches but vary in yield

• Fixation considerations:

  • For immunostaining, paraformaldehyde concentration affects epitope accessibility

  • Methanol fixation may preserve some epitopes better than PFA

  • Over-fixation can mask epitopes

• Storage effects:

  • Flash freezing samples preserves modifications

  • Avoid repeated freeze-thaw cycles

  • Protease and HDAC inhibitors prevent degradation and modification loss

• Western blot considerations:

  • SDS concentration affects histone migration

  • Transfer efficiency for small proteins requires optimization

  • PVDF membranes often perform better than nitrocellulose for histones

• ChIP-specific factors:

  • Crosslinking time affects DNA recovery and epitope accessibility

  • Sonication conditions influence fragmentation and epitope integrity

  • Buffer compositions impact antibody binding efficiency

How should Propionyl-HIST1H4A (K5) antibody be stored to maintain optimal activity?

Proper storage is essential for antibody performance:

Critical guidelines:

• Aliquot antibodies upon receipt to avoid repeated freeze-thaw cycles

• Each freeze-thaw cycle can reduce antibody activity by up to 50%

• Keep antibodies away from light, especially if conjugated

• Avoid contamination by using sterile technique

• Document thaw dates and number of freeze-thaw cycles

• Follow manufacturer-specific recommendations

According to search result #10: "Maintain refrigerated at 2-8°C for up to 2 weeks. For long term storage store at -20°C in small aliquots to prevent freeze-thaw cycles."