Propionyl-HIST1H4A (K8) Antibody

What is Propionyl-HIST1H4A (K8) Antibody and what specific modification does it recognize?

Propionyl-HIST1H4A (K8) Antibody is a primary antibody that specifically recognizes histone H4 protein that has been propionylated at lysine 8 (K8). This post-translational modification is part of the histone code that regulates chromatin structure and gene expression. The antibody targets the peptide sequence surrounding the propionylated lysine 8 position in human Histone H4 and enables researchers to study this specific epigenetic modification .

Unlike general Histone H4 antibodies, this specific antibody allows researchers to distinguish propionylation at K8 from other modifications like acetylation or methylation at the same position. The antibody is typically generated in rabbits (for polyclonal versions) and can be used across multiple experimental applications including Western blotting, immunofluorescence, chromatin immunoprecipitation (ChIP), and ELISA .

How does Propionyl-HIST1H4A (K8) modification differ from other histone H4 modifications?

Histone H4 can undergo various post-translational modifications at different residues, with propionylation at K8 being one specific type. To understand its uniqueness, we can compare it with other common modifications:

Propionylation at K8 is considered distinct from acetylation, though both are acylation modifications. While acetylation at H4K8 is well-characterized and associated with active transcription , propionylation may represent a unique regulatory mechanism potentially linked to cellular metabolism. The chemical difference between these modifications (propionyl group is larger than acetyl group) may result in distinct functional outcomes and recognition by different reader proteins.

What experimental applications is Propionyl-HIST1H4A (K8) Antibody validated for?

The Propionyl-HIST1H4A (K8) Antibody has been validated for multiple research applications:

• Enzyme-Linked Immunosorbent Assay (ELISA) - For quantitative detection of propionylated H4K8 in purified samples

• Western Blotting (WB) - For detecting the presence and relative abundance of propionylated H4K8 in cell or tissue lysates

• Immunofluorescence (IF) - For visualizing the nuclear localization and distribution patterns of propionylated H4K8

• Immunoprecipitation (IP) - For isolating propionylated H4K8-containing complexes

• Chromatin Immunoprecipitation (ChIP) - For identifying genomic regions associated with propionylated H4K8

The methodological approach for each application must be optimized based on the specific antibody characteristics. For Western blotting applications, a typical working dilution might be 1:500 to 1:1000 . For immunofluorescence, proper fixation is critical to preserve nuclear architecture while maintaining epitope accessibility. ChIP applications require careful optimization of chromatin fragmentation and antibody binding conditions to ensure specific enrichment of propionylated H4K8-associated genomic regions.

How should I design ChIP experiments using Propionyl-HIST1H4A (K8) Antibody?

Designing effective ChIP experiments with Propionyl-HIST1H4A (K8) Antibody requires careful planning and optimization:

• Sample Preparation:

  • Crosslink cells with 1% formaldehyde for 10 minutes at room temperature to preserve protein-DNA interactions

  • Quench with 125mM glycine for 5 minutes

  • Lyse cells and isolate nuclei using appropriate buffers

  • Sonicate chromatin to generate fragments of 200-500bp (optimize sonication conditions for your cell type)

• Immunoprecipitation:

  • Pre-clear chromatin with protein A/G beads

  • Incubate 2-5μg of Propionyl-HIST1H4A (K8) Antibody with chromatin overnight at 4°C

  • Include appropriate controls: IgG negative control and a positive control antibody

  • Add protein A/G beads and incubate for 2-3 hours

  • Wash thoroughly to remove non-specific binding

• DNA Recovery and Analysis:

  • Reverse crosslinks and purify DNA

  • Analyze by qPCR targeting regions of interest or perform sequencing (ChIP-seq)

Critical validation is necessary to confirm antibody specificity. Research using H4K8 modification antibodies has shown enrichment around transcription start sites , so include these regions in your initial validation experiments. For ChIP-seq analysis, consider using established peak-calling algorithms and create genome browser tracks to visualize the distribution of propionylated H4K8 across the genome.

What controls should I include when using Propionyl-HIST1H4A (K8) Antibody in immunofluorescence studies?

When performing immunofluorescence with Propionyl-HIST1H4A (K8) Antibody, include these essential controls:

• Primary Antibody Controls:

  • Negative control: Omit primary antibody but include all other steps

  • Isotype control: Use non-specific IgG from the same species (rabbit)

  • Peptide competition: Pre-incubate antibody with propionylated H4K8 peptide to confirm specificity

  • Non-propionylated control: Use cells treated with propionylation inhibitors to confirm signal specificity

• Sample Controls:

  • Positive control: Include cell types known to have high levels of H4K8 propionylation

  • Treatment comparison: Compare cells before and after treatments known to affect histone propionylation (e.g., propionyl-CoA modulators)

• Technical Controls:

  • Autofluorescence control: Examine unstained samples to detect any natural fluorescence

  • Secondary antibody control: Omit primary antibody to check for non-specific binding of secondary antibody

Document the pattern of nuclear staining, which should be consistent with chromatin localization. Based on studies of histone modifications, you might expect certain patterns of nuclear distribution that correlate with chromatin states. Typical working dilutions for immunofluorescence might range from 1:100 to 1:500, but optimization is necessary for each specific antibody preparation .

How can I optimize Western blot protocols for detecting Propionyl-HIST1H4A (K8)?

Optimizing Western blot protocols for Propionyl-HIST1H4A (K8) detection requires attention to several specific factors:

• Sample Preparation:

  • Extract histones using specialized acid extraction methods (e.g., 0.2N HCl extraction)

  • Include deacetylase and propionylation inhibitors in lysis buffers

  • Use freshly prepared samples when possible, as modifications can be lost during storage

• Gel Electrophoresis:

  • Use high percentage (15-18%) SDS-PAGE gels to resolve the low molecular weight histone proteins

  • Consider using Triton-Acid-Urea (TAU) gels for better separation of modified histones

  • Load appropriate amount of histone sample (typically 5-15μg of acid-extracted histones)

• Transfer and Detection:

  • Use PVDF membrane with 0.2μm pore size (rather than 0.45μm) for small proteins

  • Optimize transfer conditions: typically 30V overnight at 4°C works well for histones

  • Block with 5% BSA rather than milk (which contains proteins that may cross-react)

  • Use antibody at recommended dilution (typically 1:500 to 1:1000)

  • Include a loading control (total H4 or H3)

• Validation Steps:

  • Run a peptide competition control

  • Include samples with known levels of H4K8 propionylation

Expected results: Histone H4 is approximately 11kDa in size . The Propionyl-HIST1H4A (K8) Antibody should detect a single band at this molecular weight, with intensity varying based on the degree of propionylation at K8 in your samples.

How can I differentiate between propionylation and acetylation at H4K8 in my experimental data?

Differentiating between propionylation and acetylation at H4K8 is challenging due to their similar chemical structures. Here's a methodological approach:

• Antibody Specificity Testing:

  • Perform peptide competition assays using propionylated and acetylated H4K8 peptides

  • Test cross-reactivity using dot blots with modified peptides

  • Use modified histone arrays containing different modifications

• Mass Spectrometry Validation:

  • Employ LC-MS/MS to confirm the presence of propionylation vs. acetylation

  • Look for mass shifts: propionylation adds 56 Da, while acetylation adds 42 Da

  • Use targeted MS approaches to quantify relative abundance of each modification

• Functional Validation:

  • Manipulate cellular propionyl-CoA and acetyl-CoA levels to shift the modification balance

  • Examine differential enzyme sensitivity: some histone deacetylases might not remove propionyl groups

  • Use specific enzymatic inhibitors to modulate each modification separately

What are the expected genomic distribution patterns of Propionyl-HIST1H4A (K8) based on ChIP-seq data?

Based on studies of histone H4 modifications, we can infer some expected genomic distribution patterns for Propionyl-HIST1H4A (K8):

• Promoter and Enhancer Regions:

  • Similar to H4K8 acetylation, propionylation is likely enriched around transcription start sites (TSS)

  • Based on evidence that acetylation of H4K8 is enriched around TSS, propionylation may follow similar patterns

  • Expect possible enrichment at enhancer regions, particularly those active in metabolic regulation

• Gene Bodies:

  • May show differential patterns compared to acetylation

  • Possibly enriched in the gene bodies of metabolically regulated genes

  • Could correlate with exonic rather than intronic regions in actively transcribed genes

• Correlation with Chromatin States:

  • Likely associated with euchromatin (open chromatin regions)

  • May correlate with other active marks like H3K4me3 and H3K27ac

  • Potentially absent or depleted in heterochromatic regions marked by H3K9me3 or H3K27me3

• Analysis Methodology:

  • Perform peak calling using MACS2 or similar algorithms

  • Generate heatmaps and aggregate plots centered on TSS

  • Compare distribution with other histone marks using correlation analysis

  • Perform Gene Ontology enrichment analysis on propionylation-associated genes

ChIP-seq analysis has shown that acetylation of H4K8 and H4K16 are enriched around transcription start sites , and propionylation may exhibit similar or distinct patterns depending on its functional role in transcriptional regulation.

How should I integrate Propionyl-HIST1H4A (K8) ChIP-seq data with transcriptome data?

Integrating ChIP-seq data for Propionyl-HIST1H4A (K8) with transcriptome data requires a systematic approach:

• Experimental Design Considerations:

  • Collect matched samples for ChIP-seq and RNA-seq from the same experimental conditions

  • Include appropriate controls for both assays

  • Consider time course experiments to capture dynamic relationships

• Data Processing Workflow:

  • Process ChIP-seq data: quality control, alignment, peak calling, signal normalization

  • Process RNA-seq data: quality control, alignment, quantification, differential expression analysis

  • Use consistent genome builds and annotations for both datasets

• Integration Analysis Methods:

  • Assign ChIP-seq peaks to genes (e.g., by proximity to TSS or within gene bodies)

  • Correlate propionylation signal intensity with gene expression levels

  • Classify genes based on presence/absence of propionylation and expression status

  • Perform gene set enrichment analysis on co-regulated genes

• Visualization Approaches:

  • Create scatter plots of propionylation signal vs. expression level

  • Generate genome browser tracks showing both propionylation and RNA-seq data

  • Produce heatmaps clustering genes by propionylation pattern and expression

This integrative analysis can reveal whether H4K8 propionylation serves primarily as an activating mark (like many acetylation marks) or has more complex regulatory functions. Based on studies of histone acetylation around transcription start sites , you might expect positive correlation between propionylation signal at promoters and gene expression levels, though the relationship may be more nuanced for specific gene subsets.

How can Propionyl-HIST1H4A (K8) Antibody be used to investigate links between metabolism and epigenetic regulation?

Integrating Propionyl-HIST1H4A (K8) Antibody into metabolic epigenetics research provides a powerful approach to investigate links between cellular metabolism and gene regulation:

• Experimental Design Strategies:

  • Perform parallel ChIP-seq (with Propionyl-HIST1H4A (K8) Antibody), RNA-seq, and metabolomics on matched samples

  • Apply metabolic perturbations (e.g., propionate supplementation, propionyl-CoA synthetase modulation)

  • Include time course analyses to capture dynamic responses

  • Compare different metabolic states (fed vs. fasted, different carbon sources)

• Key Metabolic Conditions to Test:

  • Propionate supplementation (direct precursor to propionyl-CoA)

  • Branched-chain amino acid manipulation (source of propionyl-CoA)

  • Odd-chain fatty acid metabolism perturbation

  • Fasting/feeding cycles (alters metabolic flux)

  • Hypoxia (changes metabolic pathways)

• Analytical Approaches:

  • Monitor changes in global H4K8 propionylation levels via Western blotting

  • Map genomic distribution changes via ChIP-seq

  • Correlate propionylation changes with transcriptomic responses

  • Measure propionyl-CoA/acetyl-CoA ratios and correlate with propionylation

Propionylation is particularly relevant for metabolic studies because propionyl-CoA levels fluctuate with diet, microbiome activity, and metabolic state. This makes H4K8 propionylation a potential sensor that connects environmental inputs to epigenetic regulation. Using Propionyl-HIST1H4A (K8) Antibody allows researchers to track how these metabolic changes affect the epigenome.

What approaches should I use to study Propionyl-HIST1H4A (K8) in different cell types and tissues?

Studying H4K8 propionylation across different biological contexts requires tailored methodological approaches:

• Tissue and Cell Type Comparison Strategies:

  • Create a tissue panel for Western blot analysis of propionylation levels

  • Perform immunohistochemistry on tissue microarrays using optimized protocols

  • Isolate primary cells from different tissues for comparative ChIP-seq

  • Consider single-cell approaches for heterogeneous tissues

• Optimization for Challenging Samples:

  • For tissue samples: Optimize crosslinking conditions and chromatin extraction

  • For limited material: Adapt micro-ChIP protocols for Propionyl-HIST1H4A (K8) Antibody

  • For fixed clinical samples: Test epitope retrieval methods to restore antibody reactivity

  • For highly specialized cell types: Develop FACS sorting strategies before ChIP

• Comparative Analysis Framework:

  • Create standardized protocols to enable direct comparison between samples

  • Include reference cell lines in each experiment batch as technical controls

  • Develop quantitative metrics for propionylation levels

  • Consider tissue-specific expression of propionylation writers/erasers

• Functional Validation in Different Contexts:

  • Test metabolic sensitivities of propionylation in tissue-specific manner

  • Examine cell type-specific readers of propionylation

  • Assess functional consequences of propionylation disruption

Different cell types may exhibit distinct patterns of H4K8 propionylation reflecting their metabolic preferences and gene expression programs. Metabolically active tissues (liver, muscle, brain) might show higher or more dynamic propionylation patterns than quiescent tissues. The Propionyl-HIST1H4A (K8) Antibody enables mapping these tissue-specific epigenetic landscapes.

How can Propionyl-HIST1H4A (K8) Antibody be used in studies of cell differentiation and development?

Applying Propionyl-HIST1H4A (K8) Antibody in developmental biology research requires specialized approaches:

• Developmental Time Course Analysis:

  • Track propionylation changes during differentiation processes

  • Compare propionylation patterns across embryonic stages

  • Analyze different cell lineages as they diverge from progenitor cells

  • Correlate with expression of developmental regulators

• Cell Type-Specific Profiling:

  • Combine with cell sorting to isolate specific progenitor or differentiated populations

  • Use single-cell approaches to capture heterogeneity in propionylation

  • Perform ChIP-seq with Propionyl-HIST1H4A (K8) Antibody across distinct cell types

  • Correlate propionylation patterns with cell fate decisions

• Functional Studies:

  • Modulate propionylation during critical developmental windows

  • Assess effects on lineage commitment and differentiation potential

  • Test differentiation capacity after inhibiting propionylation writers/erasers

  • Create reporter systems to monitor propionylation dynamics in real-time

During development, cellular metabolism undergoes significant changes that may affect propionyl-CoA availability and consequently H4K8 propionylation. These changes could contribute to cell fate decisions and lineage commitment through epigenetic mechanisms. The Propionyl-HIST1H4A (K8) Antibody provides a tool to investigate these connections between metabolic state changes and epigenetic reprogramming during development.

What are common challenges when using Propionyl-HIST1H4A (K8) Antibody and how can they be addressed?

Researchers working with Propionyl-HIST1H4A (K8) Antibody may encounter several challenges:

• Specificity Issues:

  • Challenge: Cross-reactivity with acetylated H4K8 due to structural similarity

  • Solution: Perform peptide competition assays with both modifications; optimize antibody concentration; confirm with mass spectrometry

• Signal Strength Problems:

  • Challenge: Weak signal due to low abundance of propionylation

  • Solution: Increase antibody concentration; optimize incubation conditions; use signal amplification methods; enrich for histones before analysis

• Background and Non-specific Binding:

  • Challenge: High background in immunofluorescence or Western blots

  • Solution: Optimize blocking (use BSA instead of milk) ; increase washing stringency; pre-absorb antibody; titrate antibody concentration

• Sample Preparation Issues:

  • Challenge: Loss of modification during extraction

  • Solution: Include propionylation inhibitors in lysis buffers; use fresh samples; optimize extraction protocols to preserve modifications

How should I validate the specificity of my Propionyl-HIST1H4A (K8) Antibody?

Thorough validation of Propionyl-HIST1H4A (K8) Antibody specificity is critical for reliable research findings:

• Peptide Competition Assays:

  • Test antibody binding with and without pre-incubation with propionylated H4K8 peptide

  • Include acetylated H4K8 peptide to test cross-reactivity

  • Use unmodified H4K8 peptide as a negative control

  • Analyze in Western blot, ELISA, or dot blot format

• Modified Histone Panel Testing:

  • Test antibody against recombinant histones with defined modifications

  • Include H4 with various modifications (propionylation, acetylation, butyrylation at K8)

  • Test H4 with propionylation at other lysine residues (K5, K12, K16)

  • Test for sensitivity to neighboring modifications

• Genetic/Enzymatic Validation:

  • Compare samples with knockdown/knockout of propionylation writers

  • Test samples treated with propionylation inhibitors

  • Use in vitro enzymatic assays to create or remove propionylation

  • Validate with mass spectrometry as an orthogonal method

• Application-Specific Validation:

  • For ChIP: Perform sequential ChIP with general H4 antibody

  • For IF: Include peptide-blocking controls directly on slides

  • For WB: Include multiple controls and ladder of modified recombinant proteins

What are emerging alternatives to antibody-based detection of histone propionylation?

Understanding the limitations of current antibody-based approaches and exploring alternatives is important for advancing research on histone propionylation:

• Current Antibody Limitations:

  • Potential cross-reactivity with similar modifications (especially acetylation)

  • Batch-to-batch variability affecting reproducibility

  • Limited ability to detect combinatorial modifications on the same histone tail

  • Qualitative rather than truly quantitative results

• Mass Spectrometry Approaches:

  • Bottom-up, middle-down, and top-down proteomics for histone analysis

  • Targeted MS approaches for specific modification quantification

  • Advantages: Can detect combinatorial modifications; more quantitative

  • Disadvantages: Requires specialized equipment; limited spatial information

• Chemical Biology Methods:

  • Chemical probes for specific acyl modifications

  • Click chemistry approaches for modification labeling

  • Advantages: Can track dynamics in living cells; less dependent on antibody quality

  • Disadvantages: May require genetic engineering; potential off-target effects

• Next-Generation Sequencing Based Methods:

  • CUT&RUN or CUT&Tag as alternatives to traditional ChIP

  • Advantages: Requires less material; potentially higher resolution

  • Disadvantages: Still relies on antibody specificity; more complex workflow

While antibodies remain valuable tools, especially for applications like ChIP and immunofluorescence, researchers should consider complementary approaches when designing experiments to study histone propionylation. The ideal strategy often combines multiple technologies to overcome the limitations of any single approach.