Propionyl-HIST1H3A (K23) Antibody

What is the Propionyl-HIST1H3A (K23) Antibody and what biological function does it detect?

The Propionyl-HIST1H3A (K23) Antibody specifically recognizes histone H3 when it has been propionylated at lysine 23. This post-translational modification occurs on histone H3, which is a core component of nucleosomes. Nucleosomes wrap and compact DNA into chromatin, limiting DNA accessibility to cellular machineries that require DNA as a template. Histones play central roles in transcription regulation, DNA repair, DNA replication, and chromosomal stability. The propionylation at K23 is part of the complex set of post-translational modifications that regulate DNA accessibility, collectively known as the histone code .

What applications are Propionyl-HIST1H3A (K23) antibodies compatible with?

Propionyl-HIST1H3A (K23) antibodies are versatile reagents compatible with multiple experimental applications:

The specific Propionyl-HIST1H3A (K23) antibody formulation should be selected based on the intended application and experimental design .

What species reactivity is expected with Propionyl-HIST1H3A (K23) antibodies?

Propionyl-HIST1H3A (K23) antibodies demonstrate varying species reactivity depending on the specific antibody clone and manufacturer. Based on available data:

How should I optimize ChIP experiments using Propionyl-HIST1H3A (K23) antibodies?

When designing Chromatin Immunoprecipitation (ChIP) experiments with Propionyl-HIST1H3A (K23) antibodies, consider these critical optimization steps:

What factors might influence the detection specificity of Propionyl-HIST1H3A (K23) antibodies?

Several factors can impact the detection specificity of Propionyl-HIST1H3A (K23) antibodies:

• Adjacent modifications: Neighboring post-translational modifications (PTMs) may influence antibody binding. For example, acetylation or methylation at nearby residues (K18, K27) can interfere with antibody recognition of propionylation at K23.

• Epitope masking: Protein-protein interactions or chromatin compaction may prevent antibody access to the propionylated lysine.

• Cross-reactivity: Some antibodies may cross-react with acetylated H3K23, as the modifications are structurally similar. Verify antibody specificity using peptide competition assays or modified peptide arrays.

• Antibody clone: Different clones (monoclonal vs. polyclonal) may have different specificities. Recombinant monoclonal antibodies generally offer higher specificity than polyclonal antibodies.

• Sample preparation: Fixation methods and buffer compositions can influence epitope availability and antibody binding efficiency .

How can I distinguish between propionylation and other similar acylation modifications on H3K23?

Distinguishing between different acylation modifications (propionylation, acetylation, butyrylation, etc.) at H3K23 requires careful experimental design:

• Mass spectrometry validation: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) provides definitive identification of specific acyl modifications based on mass differences. Propionylation adds a mass of 56 Da, whereas acetylation adds 42 Da.

• Antibody specificity testing: Use peptide competition assays with synthetic peptides containing different acyl modifications to confirm antibody specificity:

  • H3K23pr (propionylated)

  • H3K23ac (acetylated)

  • H3K23bu (butyrylated)

• Modified peptide arrays: Test antibody binding against arrays containing various histone modifications to quantify cross-reactivity.

• Western blot comparison: Run parallel western blots with specific antibodies against different H3K23 modifications to compare their patterns.

• Enzymatic treatment: Utilize specific deacylases (such as sirtuins with preference for different acyl chains) to selectively remove specific modifications before antibody detection .

What is the relationship between H3K23 propionylation and transcriptional regulation?

H3K23 propionylation contributes to transcriptional regulation through several mechanisms:

• Chromatin structure alteration: Propionylation neutralizes the positive charge of lysine residues, potentially weakening histone-DNA interactions and promoting a more open chromatin configuration conducive to transcription.

• Reader protein recruitment: Specific reader proteins may recognize H3K23pr and recruit transcriptional machinery. Current research suggests propionylation may be recognized by proteins containing bromodomains or YEATS domains.

• Genomic distribution: ChIP-seq studies indicate H3K23pr is often enriched at promoters and enhancers of actively transcribed genes, suggesting a positive role in gene expression.

• Metabolic sensing: Propionylation levels may reflect cellular propionyl-CoA abundance, potentially linking metabolic state to gene expression changes.

• Interplay with other modifications: H3K23pr may function cooperatively or competitively with other histone modifications like H3K23ac or methylation marks on neighboring residues to fine-tune transcriptional responses .

How do propionylation levels at H3K23 change during cellular differentiation or disease states?

Propionylation at H3K23 exhibits dynamic changes during cellular differentiation and in disease contexts:

• Developmental dynamics: During cellular differentiation, significant remodeling of the epigenetic landscape occurs. Preliminary studies suggest H3K23pr levels may change at developmental gene loci during stem cell differentiation, although comprehensive mapping across development stages remains incomplete.

• Cancer alterations: In various cancer types, abnormal histone acylation patterns, including propionylation, have been observed. These changes may contribute to dysregulated gene expression supporting tumorigenesis.

• Metabolic disorders: As propionylation depends on propionyl-CoA levels, conditions affecting propionate metabolism (such as propionic acidemia or methylmalonic acidemia) may show altered H3K23pr patterns.

• Neurodegenerative diseases: Emerging research suggests histone acylation abnormalities may contribute to neurodegenerative pathologies, though specific H3K23pr involvement requires further investigation.

• Inflammatory conditions: Changes in metabolic pathways during inflammation may influence propionyl-CoA availability and consequently affect H3K23pr levels .

What sample preparation protocols maximize detection of Propionyl-HIST1H3A (K23) modifications?

Optimal sample preparation is critical for preserving and detecting H3K23 propionylation:

• Cell harvesting and nuclear isolation:

  • Use fresh samples whenever possible

  • Include histone deacetylase inhibitors (e.g., sodium butyrate, trichostatin A) and deacylase inhibitors (e.g., nicotinamide) in buffers

  • Maintain low temperatures throughout processing to minimize enzymatic activity

• Histone extraction protocols:

  • Acid extraction (0.2N HCl or 0.4N H2SO4) efficiently isolates histones while preserving most modifications

  • Salt extraction methods may be preferable for certain applications

  • Include protease inhibitors and deacylase inhibitors in all buffers

• Fixation for immunocytochemistry/immunofluorescence:

  • Brief fixation (10 minutes) with 4% paraformaldehyde preserves epitope accessibility

  • Permeabilization with 0.1-0.5% Triton X-100 for 10 minutes typically provides good antibody access

  • Consider epitope retrieval methods if signal is weak

• Western blot considerations:

  • Use freshly prepared SDS-PAGE gels with 15-18% acrylamide for optimal histone separation

  • Transfer to PVDF membranes (rather than nitrocellulose) often improves results for histone detection

  • Block with 5% BSA rather than milk to prevent non-specific binding

How can I optimize Western blot detection of Propionyl-HIST1H3A (K23)?

For optimal Western blot detection of Propionyl-HIST1H3A (K23), follow these recommendations:

• Sample preparation:

  • Load 5-15μg of acid-extracted histones or 20-30μg of whole cell lysate

  • Include phosphatase and deacylase inhibitors during sample preparation

  • Use fresh samples when possible, as freeze-thaw cycles may affect modification stability

• Gel electrophoresis:

  • Use 15-18% acrylamide gels for optimal histone resolution

  • Consider Triton-Acid-Urea (TAU) gels for separation based on charge differences from modifications

  • Run at lower voltage (80-100V) to improve resolution

• Transfer conditions:

  • Transfer to PVDF membranes at 30V overnight at 4°C for complete transfer

  • Use transfer buffer containing 0.1% SDS to improve histone transfer

  • Verify transfer efficiency with reversible staining (Ponceau S)

• Antibody incubation:

  • Block with 5% BSA in TBST (not milk, which contains proteins that may interfere)

  • Dilute primary antibody (typically 1:500-1:1000) in 5% BSA/TBST

  • Incubate overnight at 4°C with gentle rocking

  • Extensive washing (5-6 times for 5 minutes each) improves signal-to-noise ratio

• Detection optimization:

  • Enhanced chemiluminescence (ECL) substrate with longer exposure times may be necessary

  • Consider using fluorescent secondary antibodies for more quantitative analysis

  • Always include a loading control (total H3 or H4 antibody)

What controls should be included when working with Propionyl-HIST1H3A (K23) antibodies?

Proper controls are essential for reliable results with Propionyl-HIST1H3A (K23) antibodies:

• Positive controls:

  • Cell lines known to have detectable H3K23pr levels (e.g., HeLa, HEK293)

  • Synthetic propionylated H3K23 peptides

  • Recombinant H3 propionylated in vitro using propionyl-CoA and histone acetyltransferases

• Negative controls:

  • Antibody isotype controls matched to the primary antibody

  • Peptide competition assays using excess propionylated peptide to confirm specificity

  • Samples treated with deacylases to remove propionylation

• Specificity controls:

  • Parallel testing with antibodies against other H3K23 modifications (acetylation, butyrylation)

  • Testing on H3K23 mutant constructs (K23R or K23A) that cannot be propionylated

  • Dot blots with modified and unmodified peptides at various concentrations

• Normalization controls:

  • Total H3 levels (using pan-H3 antibodies)

  • Housekeeping proteins for loading control (though less ideal than total H3)

  • For ChIP experiments, input chromatin and IgG controls are essential

• Treatment controls:

  • Histone deacetylase inhibitors (which may increase propionylation)

  • Propionate supplementation (which may increase cellular propionyl-CoA levels)

  • Genetic manipulation of enzymes involved in propionylation/depropionylation

What are common causes of weak or absent signal when using Propionyl-HIST1H3A (K23) antibodies?

When experiencing weak or absent signals with Propionyl-HIST1H3A (K23) antibodies, consider these potential issues and solutions:

• Low abundance of the modification:

  • H3K23pr may naturally occur at low levels in your sample

  • Consider treating cells with propionate or histone deacetylase inhibitors to boost levels

  • Use more sensitive detection methods (e.g., enhanced chemiluminescence substrates, longer exposure times)

• Epitope masking or destruction:

  • Improper sample preparation may destroy the modification

  • Overfixation can block antibody access to the epitope

  • Try different extraction methods or fixation protocols

  • Consider antigen retrieval techniques for fixed samples

• Antibody-related issues:

  • Antibody degradation due to improper storage or handling

  • Insufficient antibody concentration

  • Batch-to-batch variations in antibody quality

  • Try increasing antibody concentration or testing a new lot

• Technical factors:

  • Inefficient protein transfer in Western blots

  • Excessive blocking or washing

  • Incompatible buffers or reagents

  • Optimize each step of the protocol independently

• Biological variability:

  • Cell cycle-dependent fluctuations in modification levels

  • Stress or culture conditions affecting histone modifications

  • Standardize experimental conditions and timing of sample collection

How can I quantitatively analyze Propionyl-HIST1H3A (K23) modification levels across different samples?

For quantitative analysis of H3K23pr levels across samples, implement these approaches:

• Western blot quantification:

  • Use fluorescent secondary antibodies for wider linear detection range

  • Always normalize to total H3 levels from the same samples

  • Use high-quality image acquisition with exposure below saturation

  • Analyze with software like ImageJ, normalizing signal intensity to loading controls

• Mass spectrometry-based quantification:

  • Use stable isotope labeling (SILAC) for direct comparison between samples

  • Implement multiple reaction monitoring (MRM) for targeted quantification

  • Consider chemical derivatization strategies to improve detection

  • Use synthetic peptide standards for absolute quantification

• ChIP-seq analysis:

  • Normalize to input chromatin and library size

  • Use spike-in controls (e.g., Drosophila chromatin) for between-sample normalization

  • Calculate enrichment relative to background or IgG control

  • Compare propionylation levels at specific genomic regions across conditions

• Immunofluorescence quantification:

  • Maintain identical acquisition settings across all samples

  • Use nuclear counterstains to normalize signal intensity per nucleus

  • Analyze multiple fields and cells for statistical power

  • Consider flow cytometry for high-throughput quantification

• ELISA-based approaches:

  • Develop standard curves using synthetic modified peptides

  • Use consistent amounts of total histone for each sample

  • Run technical replicates to assess measurement precision

  • Include inter-assay calibrators for comparing across plates

What methods can differentiate global versus locus-specific changes in H3K23 propionylation?

To distinguish between global and locus-specific changes in H3K23 propionylation, researchers should employ a combination of techniques: