Propionyl-HIST1H4A (K31) Antibody

What is Propionyl-HIST1H4A (K31) and why is it significant in epigenetic research?

Propionyl-HIST1H4A (K31) refers to the propionylation (addition of a propionyl group) at lysine residue 31 of histone H4, one of the core components of nucleosomes. This post-translational modification represents one of several novel acylation marks discovered alongside butyrylation . The propionylation of lysine residues neutralizes the positive charge of lysine, which can significantly impact protein folding and function .

Histone H4 is involved in chromatin structure and function, playing crucial roles in DNA packaging and gene regulation . The modification at K31 specifically contributes to the complex epigenetic code that governs various cellular processes including transcriptional regulation, DNA repair, and replication. Understanding site-specific propionylation provides researchers with insights into the nuanced mechanisms of chromatin-based gene regulation.

How does propionylation differ from acetylation in terms of chemical structure and biological function?

Propionylation involves the addition of a propionyl group (CH₃CH₂CO-) to the ε-amino group of lysine residues, while acetylation involves the addition of an acetyl group (CH₃CO-). The propionyl group contains one additional carbon compared to acetyl, making it slightly larger and more hydrophobic . This structural difference, though subtle, may create distinct binding surfaces for reader proteins that recognize these modifications.

Both modifications neutralize the positive charge of lysine residues and can be catalyzed by similar enzymes, including p300/CBP . Interestingly, research has demonstrated that many lysine residues that can be acetylated in histone H4 (including K5, K8, K12, K16, and K31) can also be propionylated . This suggests a potential metabolic regulation of the histone code where the availability of acetyl-CoA versus propionyl-CoA could influence which modification occurs.

What validated applications exist for Propionyl-HIST1H4A (K31) Antibody?

The Propionyl-HIST1H4A (K31) Polyclonal Antibody has been validated for several research applications:

• Western Blotting (WB): For detection of propionylated H4K31 in protein extracts, typically showing a band at approximately 14 kDa corresponding to histone H4

• Chromatin Immunoprecipitation (ChIP): For investigating the genomic distribution of propionylated H4K31 and its association with specific DNA regions

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection of propionylation at K31 of histone H4

These applications enable comprehensive analysis of propionylation from the protein level (Western blot) to genome-wide mapping (ChIP). The antibody has been specifically validated for human samples, though researchers should conduct preliminary tests when applying it to other species .

How should researchers optimize experimental conditions for detecting propionylation using Western blot analysis?

Western blot detection of propionylated histone H4 at K31 requires careful optimization:

Sample Preparation:

• Extract histones using acid extraction methods (0.2N HCl or 0.4N H₂SO₄) or commercial histone extraction kits

• Include histone deacetylase inhibitors (e.g., sodium butyrate at 10mM) in buffers to preserve modifications

• Add protease inhibitors to prevent degradation

• Maintain low temperature throughout the preparation process

Western Blot Protocol:

• Use high-percentage (15-18%) SDS-PAGE gels for optimal resolution of low molecular weight histone proteins

• Load 10-20 μg of histone extract per lane

• Transfer to PVDF membrane rather than nitrocellulose for better protein retention

• Block with 5% BSA rather than milk (which contains histones and can increase background)

• Use the Propionyl-HIST1H4A (K31) antibody at 0.1-1 μg/mL concentration

Controls and Validation:

• Include a positive control (cells treated with sodium butyrate, which can enhance propionylation)

• Include a negative control (samples where propionylation is expected to be absent)

• Confirm specificity using a total histone H4 antibody on parallel blots

• Consider peptide competition assays to validate specificity of the signal

For optimal detection, use HRP-conjugated secondary antibodies with enhanced chemiluminescence, expecting a specific band at approximately 14 kDa for histone H4 .

What are best practices for Chromatin Immunoprecipitation (ChIP) experiments using Propionyl-HIST1H4A (K31) Antibody?

For effective ChIP experiments with Propionyl-HIST1H4A (K31) Antibody:

Chromatin Preparation:

• Cross-link cells with 1% formaldehyde for 10 minutes at room temperature

• Quench with 125 mM glycine for 5 minutes

• Isolate nuclei and sonicate chromatin to achieve fragments of 200-500 bp

• Pre-clear chromatin with protein G beads to reduce background

Immunoprecipitation:

• Use 2-5 μg of Propionyl-HIST1H4A (K31) Antibody per reaction

• Incubate with chromatin overnight at 4°C

• Add protein G beads and incubate for 2-4 hours

• Perform stringent washing steps to remove non-specific binding

• Elute chromatin-antibody complexes and reverse cross-links

• Purify DNA for downstream analysis (qPCR or sequencing)

Critical Controls:

• Input chromatin (non-immunoprecipitated) - typically 5-10% of starting material

• IgG control - using rabbit IgG to match the host species of the primary antibody

• Positive control - using an antibody against total histone H4

• Known genomic regions where propionylation is expected/not expected

For ChIP-seq applications, additional quality control steps should be implemented to ensure library complexity and sufficient sequencing depth. Researchers should consider the distribution pattern of propionylation marks when designing analysis pipelines.

How can mass spectrometry be used to validate and extend antibody-based detection of propionylation?

Mass spectrometry (MS) offers complementary approaches to antibody-based detection of propionylation:

Sample Preparation for MS:

• Isolate histones using acid extraction

• Digest with appropriate proteases (trypsin yields predictable fragments for histone H4)

• Consider enrichment strategies for modified peptides

• For targeted analysis, develop specific MS methods for the K31-containing peptide

MS Analysis Approaches:

• Shotgun proteomics to identify propionylated peptides in complex mixtures

• Targeted MS (PRM/MRM) for quantitative analysis of specific propionylated peptides

• Middle-down approaches to preserve information about co-occurring modifications

• Time-lapse enzymatic deacetylation to distinguish between acetylation and propionylation

Data Analysis Considerations:

• Search MS data with propionylation (+56.026 Da) as a variable modification

• Apply strict false discovery rate controls to ensure confidence in identifications

• Use high mass accuracy to distinguish propionylation from other modifications

• Verify site localization using appropriate scoring algorithms

• Normalize data against unmodified peptides for quantitative comparisons

Integration with Antibody-Based Methods:

• Use MS to verify specificity of antibody by analyzing immunoprecipitated material

• Compare relative abundances of modifications detected by both methods

• Combine ChIP-seq distribution data with MS quantification for comprehensive analysis

This integrated approach provides both site-specific quantification and genome-wide distribution information, offering a more complete picture of propionylation biology.

How can researchers distinguish between propionylation and other similar lysine modifications in experimental data?

Distinguishing propionylation from similar modifications like acetylation and butyrylation requires multi-faceted approaches:

By Mass Spectrometry:

• High-resolution MS can differentiate based on precise mass differences:

  • Acetylation: +42.011 Da

  • Propionylation: +56.026 Da

  • Butyrylation: +70.042 Da

• Diagnostic fragment ions in MS/MS spectra can provide modification-specific signatures

• Chromatographic behavior - propionylated peptides generally elute later than acetylated peptides due to increased hydrophobicity

• Enzymatic treatments with specific deacylases that preferentially remove certain modifications

By Antibody-Based Methods:

• Peptide competition assays using synthetic peptides with different modifications

• Sequential immunoprecipitation with antibodies specific to different modifications

• Parallel detection with modification-specific antibodies

• Western blot analysis following treatments that selectively affect specific modifications

Validation Strategies:

• Cross-validate findings between MS and antibody-based methods

• Compare results with published datasets on modification distribution

• Use genetic or chemical manipulations to alter specific modification pathways

• Apply the time-lapse enzymatic approach to monitor differential removal of modifications

Careful attention to these approaches helps researchers accurately identify propionylation events and distinguish them from similar modifications with high confidence.

What are the key considerations for analyzing ChIP-seq data for propionylated histones?

Analyzing ChIP-seq data for propionylated histones requires special considerations:

Quality Control:

• Evaluate enrichment metrics (fraction of reads in peaks, FRiP)

• Assess library complexity and duplication rates

• Compare signal-to-noise ratios across replicates

• Examine peak profiles at known control regions

Peak Calling Strategy:

• Use appropriate peak callers (MACS2 is commonly used)

• Consider broader peak profiles characteristic of histone modifications

• Compare with input controls and IgG controls to identify true enrichment

• Implement suitable false discovery rate thresholds

Comparative Analysis:

• Compare propionylation patterns with other histone modifications

• Correlate with gene expression data to identify functional associations

• Analyze overlap with chromatin states (enhancers, promoters, etc.)

• Examine cell type-specific versus conserved propionylation patterns

Functional Interpretation:

• Perform gene ontology enrichment of propionylated regions

• Identify transcription factor binding motifs enriched in propionylated regions

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

• Correlate with three-dimensional chromatin organization data

Visualization and Reporting:

• Generate genome browser tracks showing propionylation profiles

• Create heatmaps centered on genomic features (TSS, enhancers)

• Use aggregation plots to show average profiles across feature classes

• Compare replicates to demonstrate reproducibility

These analytical approaches help researchers extract meaningful biological insights from propionylation ChIP-seq data, revealing potential functions of this modification in chromatin regulation.

How does propionylation at K31 interact with other histone modifications in the histone code?

Propionylation at K31 of histone H4 exists within a complex network of histone modifications:

Co-occurrence Patterns:

• Propionylation at K31 can co-exist with modifications at other residues on histone H4

• Research has shown that propionylation can occur simultaneously at multiple lysine residues, including K5, K8, K12, K16, K31, K44, K77, K79, and K91 of histone H4

• Certain modifications may be mutually exclusive with K31 propionylation due to structural constraints

Enzymatic Regulation:

• The same enzymes that catalyze acetylation, such as p300/CBP, can also mediate propionylation

• This creates a potential regulatory mechanism where cellular metabolism (acetyl-CoA vs. propionyl-CoA availability) may influence modification patterns

• The specificity of deacylases for propionylated lysines may differ from their specificity for acetylated lysines

Functional Implications:

• Propionylation at K31 may work in concert with other modifications to establish specific chromatin states

• The combination of modifications likely creates distinct binding surfaces for reader proteins

• These combinatorial patterns may direct specific transcriptional responses or chromatin remodeling events

Methodological Approaches to Study Interactions:

• Sequential ChIP (re-ChIP) to identify co-occurring modifications

• Mass spectrometry of intact histone tails to preserve modification combinations

• Correlative analysis of ChIP-seq datasets for different modifications

• Genetic or chemical perturbation of specific modifications to observe effects on others

Understanding these interactions is crucial for deciphering the complete functional role of propionylation in chromatin biology and gene regulation.

What roles does propionylation play in cellular metabolism and disease processes?

Propionylation sits at the intersection of metabolism and epigenetic regulation with important implications for disease:

Metabolic Connections:

• Propionyl-CoA is derived from the metabolism of odd-chain fatty acids, certain amino acids (valine, isoleucine, methionine, threonine), and cholesterol

• Changes in cellular metabolism can alter propionyl-CoA levels, potentially affecting histone propionylation patterns

• The propionyl-CoA to acetyl-CoA ratio may serve as a metabolic sensor that influences the epigenetic landscape

Potential Disease Associations:

• Cancer: Altered metabolism in cancer cells may affect propionylation patterns, contributing to dysregulated gene expression

• Metabolic disorders: Conditions affecting propionate metabolism could impact histone propionylation

• Neurodegenerative diseases: Epigenetic dysregulation, including abnormal histone modifications, has been implicated in neurodegeneration

• Inflammatory conditions: Histone modifications regulate inflammatory gene expression

Research Approaches:

• Compare propionylation patterns between normal and disease tissues

• Examine effects of metabolic perturbations on global propionylation levels

• Investigate genetic variants in enzymes that regulate propionylation

• Develop small molecules that specifically target propionylation/depropionylation

Therapeutic Implications:

• Targeting the enzymes that regulate propionylation could represent a novel therapeutic approach

• Dietary interventions that alter propionyl-CoA levels might modulate epigenetic states

• Biomarkers based on propionylation patterns may have diagnostic or prognostic value

This emerging area of research connects fundamental biochemistry with disease mechanisms, offering new perspectives on metabolic regulation of gene expression.

What emerging technologies are advancing our understanding of site-specific histone propionylation?

Several cutting-edge technologies are enhancing our ability to study histone propionylation:

Advanced Mass Spectrometry:

• Top-down proteomics approaches for analyzing intact histone proteins

• Middle-down methods using limited proteolysis to generate larger histone fragments

• Targeted quantitative MS using parallel reaction monitoring (PRM) for sensitive quantification

• Time-lapse enzymatic deacetylation coupled with MS to distinguish between modifications

• Cross-linking MS to identify proteins that interact with propionylated histones

Genomic Mapping Innovations:

• CUT&RUN or CUT&Tag methods offering higher resolution and lower background than traditional ChIP

• Single-cell ChIP-seq to reveal cell-to-cell variability in propionylation patterns

• ChIP-STARR-seq to assess the functional impact of propionylation on enhancer activity

• Long-read sequencing to map propionylation across extended genomic regions

Genetic Engineering:

• CRISPR-based approaches to mutate specific lysine residues

• Engineered reader domains to detect specific modifications

• Optogenetic control of enzymes that add or remove propionyl groups

• Synthetic histone systems with defined modification patterns

Structural Biology:

• Cryo-EM studies of nucleosomes containing propionylated histones

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

• Hydrogen-deuterium exchange mass spectrometry to study structural dynamics

• Molecular dynamics simulations to predict the impact of propionylation on chromatin structure

Integrative Approaches:

• Multi-omics integration combining ChIP-seq, RNA-seq, and metabolomics

• Machine learning algorithms to predict propionylation sites and functional outcomes

• Systems biology modeling of the interplay between metabolism and histone modifications

These technologies are expanding our ability to study propionylation with unprecedented resolution, enabling deeper insights into its functional roles in diverse biological processes.

What are the most common technical challenges when working with Propionyl-HIST1H4A (K31) Antibody?

Researchers commonly encounter several technical challenges when working with Propionyl-HIST1H4A (K31) Antibody:

Specificity Issues:

• Cross-reactivity with other histone modifications (particularly acetylation and butyrylation)

• Potential recognition of propionylation at other lysine residues in histones

• Non-specific binding to other proteins in complex samples

Sensitivity Limitations:

• Low abundance of propionylation marks in certain cell types or conditions

• Signal-to-noise challenges in ChIP experiments

• Detection limits in Western blotting applications

Sample Preparation Challenges:

• Loss of modifications during extraction and processing

• Artificial introduction of modifications during sample handling

• Inconsistent histone extraction efficiency between samples

• Inadequate chromatin fragmentation for ChIP applications

Experimental Design Considerations:

• Selection of appropriate positive and negative controls

• Determining optimal antibody concentration for each application

• Batch effects between experiments affecting reproducibility

• Appropriate normalization strategies for quantitative comparisons

Troubleshooting Approaches:

• Validate antibody specificity using peptide competition assays

• Optimize fixation and extraction protocols to preserve modifications

• Include enzyme inhibitors to prevent modification loss

• Perform titration experiments to determine optimal antibody concentrations

• Use multiple antibody lots and replicates to ensure reproducibility

Addressing these challenges requires careful optimization and validation steps to ensure reliable and reproducible results when working with propionylation-specific antibodies.

How can researchers validate the specificity of Propionyl-HIST1H4A (K31) Antibody?

Validating antibody specificity is crucial for reliable propionylation research:

Peptide-Based Validation:

• Peptide competition assays using propionylated and unmodified K31 peptides

• Dot blots with synthetic peptides containing different modifications (acetylation, propionylation, butyrylation) at K31

• ELISA assays using modification-specific peptide arrays

• Testing against peptides with propionylation at other lysine residues in histone H4

Cellular and Biochemical Validation:

• Western blot analysis of samples with enzymatically increased or decreased propionylation

• Comparison of signal in wild-type cells versus cells with K31R mutation (if available)

• IP-Western experiments to confirm specificity of immunoprecipitated material

• Mass spectrometry analysis of immunoprecipitated histones to confirm modification status

Experimental Controls:

• Positive controls: Cells treated with sodium butyrate or propionate to increase propionylation

• Negative controls: Samples treated with deacylases to remove modifications

• Specificity controls: Parallel detection with antibodies against other modifications

• Technical controls: Secondary antibody-only controls to assess background

Orthogonal Validation:

• Correlation of antibody-based results with mass spectrometry data

• Comparison with other commercially available antibodies targeting the same modification

• Functional validation through perturbation studies

• Reproducibility across multiple experimental systems and conditions

Documentation and Reporting:

• Maintain detailed records of validation experiments

• Document antibody lot information and variations in performance

• Report validation methods in publications

• Share validation data with the scientific community

These comprehensive validation strategies help ensure that research findings based on Propionyl-HIST1H4A (K31) Antibody accurately reflect the biology of histone propionylation rather than technical artifacts.

What are the emerging questions in the field of histone propionylation research?

The study of histone propionylation is evolving rapidly, with several key questions driving future research:

Regulatory Mechanisms:

• What is the complete enzymatic machinery responsible for adding and removing propionyl groups?

• How is site-specificity achieved in propionylation reactions?

• What is the interplay between metabolism and propionylation dynamics?

• How do cells regulate the balance between different acylation types (acetylation, propionylation, butyrylation)?

Functional Consequences:

• What are the specific reader proteins for propionylated histones?

• How does propionylation at K31 specifically affect chromatin structure and gene expression?

• What is the evolutionary conservation of propionylation patterns across species?

• How do propionylation patterns change during development and cellular differentiation?

Disease Relevance:

• Are propionylation patterns altered in specific disease states?

• Can propionylation serve as a biomarker for metabolic disorders or cancer?

• Is targeted modulation of propionylation a viable therapeutic strategy?

• How do environmental factors and diet influence global propionylation levels?

Technological Innovations:

• How can we develop more specific tools to distinguish between closely related modifications?

• What approaches can provide single-cell resolution of propionylation patterns?

• Can computational models predict functional outcomes of propionylation changes?

• How can we achieve site-specific manipulation of propionylation in living cells?

These questions represent exciting frontiers in epigenetic research, with potential implications for understanding fundamental biology and developing new therapeutic approaches.

What methodological improvements would advance propionylation research?

Several methodological improvements could significantly advance propionylation research:

Antibody Development:

• Generation of monoclonal antibodies with improved specificity for propionylated K31

• Development of antibodies that can distinguish between different acylation types

• Creation of antibodies recognizing specific combinations of modifications

• Standardized validation protocols for modification-specific antibodies

Mass Spectrometry Enhancements:

• Improved fragmentation methods to better localize and identify propionylation sites

• Development of targeted assays for quantifying low-abundance propionylated peptides

• Enhanced separation techniques to distinguish isomeric modified peptides

• Streamlined workflows for high-throughput analysis of histone modifications

Genetic and Chemical Tools:

• Site-specific incorporation of propionylated lysines using genetic code expansion

• Development of selective inhibitors for enzymes that add or remove propionyl groups

• Engineered reader domains for detecting specific modifications in living cells

• CRISPR-based approaches for manipulating specific lysine residues

Computational Resources:

• Enhanced database search algorithms specifically designed for histone modifications

• Machine learning tools to predict propionylation sites and functional impacts

• Integrative analysis platforms combining multi-omics data

• Standardized data repositories for histone modification datasets

Functional Assays:

• Development of high-throughput assays to assess functional consequences of propionylation

• Improved methods for studying chromatin dynamics in the context of specific modifications

• Single-molecule approaches to analyze the impact of propionylation on nucleosome behavior

• Cellular systems with controllable propionylation levels

These methodological improvements would enable more precise, sensitive, and comprehensive studies of histone propionylation, advancing our understanding of its biological roles and potential therapeutic applications.