Crotonyl-HIST1H4A (K5) Antibody

What is the Crotonyl-HIST1H4A (K5) Antibody and what biological modification does it detect?

The Crotonyl-HIST1H4A (K5) Antibody specifically recognizes the crotonylation modification at lysine 5 (K5) of histone H4. Histone H4 is a core component of nucleosomes, which wrap and compact DNA into chromatin. This modification is part of the histone code that regulates DNA accessibility to cellular machinery requiring DNA as a template . The antibody binds to the peptide sequence around the site of crotonyl-lysine at position 5 derived from human histone H4 . This post-translational modification plays a crucial role in epigenetic gene regulation, DNA repair, DNA replication, and chromosomal stability .

What applications has the Crotonyl-HIST1H4A (K5) Antibody been validated for?

The antibody has been validated for multiple research applications across various platforms:

These applications allow researchers to investigate the presence, distribution, and relative abundance of H4K5 crotonylation in different experimental contexts .

How does crotonylation differ from other histone H4 modifications such as acetylation?

Crotonylation is structurally distinct from acetylation, featuring a four-carbon chain with an α,β-unsaturated carbonyl group, compared to acetylation's single-carbon modification. This structural difference results in a more bulky modification that may create different binding surfaces for reader proteins. While acetylation at H4K5 is often associated with newly assembled histones (especially when K12 is also acetylated but K8 is unacetylated), crotonylation at the same position may have distinct functional implications in gene regulation . The distribution patterns of H4K5 crotonylation can be distinguished from other modifications using specific antibodies like the one discussed here, allowing researchers to investigate the unique biological roles of this modification .

What are the best practices for optimizing Western blot protocols with Crotonyl-HIST1H4A (K5) Antibody?

For optimal Western blot results with Crotonyl-HIST1H4A (K5) Antibody:

• Sample preparation: Extract histones using acid extraction methods to maximize histone yield and preservation of modifications.

• Gel selection: Use 15-18% SDS-PAGE gels to properly resolve the low molecular weight histone proteins (~11-15 kDa).

• Transfer conditions: Optimize transfer to PVDF membranes (preferred over nitrocellulose) using 20% methanol buffer at lower voltage (30V) for longer periods (2 hours) to ensure efficient transfer of small proteins.

• Blocking: Use 5% BSA in TBST rather than milk, as milk contains enzymes that may remove some histone modifications.

• Antibody dilution: Start with a 1:1000 dilution for polyclonal antibodies and 1:2000 for monoclonal versions, optimizing as needed .

• Validation controls: Include positive controls (cells known to exhibit H4K5 crotonylation) and negative controls (samples treated with decrotonylase enzymes) .

How should researchers design ChIP experiments using Crotonyl-HIST1H4A (K5) Antibody?

Designing effective ChIP experiments with this antibody requires careful consideration of several factors:

• Crosslinking optimization: Standard 1% formaldehyde for 10 minutes at room temperature works well for histone modifications, but optimization may be required for specific cell types.

• Sonication parameters: Aim for chromatin fragments between 200-500 bp for optimal resolution. Verify fragment size by agarose gel electrophoresis.

• Antibody amount: Use 5 μg of antibody per ChIP reaction with 4×10^6 cells as a starting point .

• Controls: Always include:

  • Input control (pre-immunoprecipitation chromatin)

  • IgG negative control (same species as the primary antibody)

  • Positive control (antibody against abundant histone mark like H3K4me3)

• Quantification: Use real-time PCR for targeted analysis or next-generation sequencing (ChIP-seq) for genome-wide profiling.

• Data analysis: For ChIP-seq, compare enrichment patterns of H4K5cr with other histone marks (H4K5ac, H3K27ac) to understand the functional significance of this modification .

What are recommended immunofluorescence protocols for visualizing nuclear distribution of H4K5 crotonylation?

For optimal immunofluorescence results:

• Fixation: Use 4% paraformaldehyde for 10 minutes at room temperature, followed by permeabilization with 0.2% Triton X-100.

• Antigen retrieval: Perform mild heat-induced epitope retrieval using 10 mM sodium citrate buffer (pH 6.0) to improve accessibility of nuclear antigens.

• Blocking: Use 5% BSA with 0.1% Triton X-100 in PBS for 1 hour at room temperature.

• Primary antibody incubation: Dilute the Crotonyl-HIST1H4A (K5) Antibody 1:100 in blocking buffer and incubate overnight at 4°C.

• Secondary antibody: Use fluorophore-conjugated secondary antibodies (e.g., Alexa Fluor 488) at 1:500 dilution for 1 hour at room temperature.

• Counterstaining: DAPI (1 μg/mL) for nuclear visualization.

• Mounting: Use anti-fade mounting medium to preserve fluorescence signal.

• Imaging: Confocal microscopy is recommended for detailed nuclear localization patterns .

How can researchers validate the specificity of Crotonyl-HIST1H4A (K5) Antibody in their experimental systems?

Validating antibody specificity is crucial for reliable interpretation of experimental results:

• Peptide competition assay: Pre-incubate the antibody with the crotonylated H4K5 peptide immunogen before application to your sample. This should abolish specific signals.

• Recombinant protein testing: Test the antibody against a panel of recombinant histones with different modifications (acetylation, methylation, crotonylation) at the K5 position and other positions to confirm specificity .

• Genetic validation: Use CRISPR/Cas9 to create cell lines with K5R mutation in histone H4 genes, which would prevent crotonylation at this position.

• Enzyme treatment: Treat samples with decrotonylases (such as certain sirtuins) and observe signal reduction.

• Cross-reactivity assessment: Test against samples containing other acylations (acetylation, butyrylation) to ensure the antibody doesn't cross-react with similar modifications .

What are the known cross-reactivity issues with Crotonyl-HIST1H4A (K5) Antibody?

Potential cross-reactivity concerns include:

• Similar acyl modifications: Some antibody preparations may show cross-reactivity with other acyl modifications at the same position (H4K5ac, H4K5bu), particularly if the immunizing peptide design didn't adequately distinguish these modifications.

• Neighboring modifications: The antibody specificity may be affected by modifications on neighboring residues. For example, some H4K5cr antibodies may have reduced binding if K8 is also modified .

• Isoform specificity: While the antibody targets HIST1H4A, the high sequence conservation among H4 variants means the antibody will recognize crotonylation at position K5 in all H4 protein variants (HIST1H4A-L, HIST2H4, HIST4H4) .

• Species cross-reactivity: The antibody raised against human H4K5cr typically works well with mouse and rat samples due to high sequence conservation, but validation is recommended when using with other species .

How can Crotonyl-HIST1H4A (K5) Antibody be used to study the dynamics of histone crotonylation during cell cycle progression?

Investigating crotonylation dynamics throughout the cell cycle:

• Synchronization methods: Use double thymidine block, nocodazole treatment, or mitotic shake-off to obtain cell populations at specific cell cycle stages.

• Multi-parameter flow cytometry: Combine propidium iodide staining for DNA content with intracellular H4K5cr antibody staining to correlate crotonylation levels with cell cycle phases.

• Time-course experiments: After synchronization release, collect cells at regular intervals (e.g., every 2 hours) and analyze H4K5cr levels by Western blot and immunofluorescence.

• ChIP-seq at different cell cycle stages: Perform ChIP-seq using the Crotonyl-HIST1H4A (K5) Antibody on synchronized populations to map genome-wide changes in crotonylation patterns.

• Comparison with other modifications: Parallel analysis of H4K5ac and H4K5cr can reveal whether these modifications show similar or distinct patterns during cell cycle progression, building on previous findings regarding H4 acetylation dynamics .

What methodological approaches can be used to study the relationship between H4K5 crotonylation and transcriptional regulation?

To investigate the functional relationship between H4K5 crotonylation and gene expression:

• Integrated genomic analysis: Combine ChIP-seq data for H4K5cr with RNA-seq data from the same biological sample to correlate genomic localization with gene expression levels.

• Sequential ChIP (Re-ChIP): Perform sequential immunoprecipitation with H4K5cr antibody followed by antibodies against transcriptional machinery components (RNA Pol II, transcription factors) to identify co-occupancy.

• CRISPR-based approaches:

  • dCas9-fusion of crotonyl readers or erasers to specific genomic loci

  • Mutation of H4K5 to arginine to prevent crotonylation

  • Targeting crotonylation regulators (writers, erasers) to study effects on target genes

• Global manipulation of crotonylation: Modulate cellular crotonyl-CoA levels through metabolic interventions or enzyme inhibition, then assess genome-wide H4K5cr patterns and corresponding transcriptional changes.

• Single-cell approaches: Combine immunofluorescence for H4K5cr with RNA-FISH for specific transcripts to correlate modification status with transcriptional output at the single-cell level .

How can site-specific incorporation of crotonyllysine be used to validate Crotonyl-HIST1H4A (K5) Antibody specificity and function?

The genetic code expansion approach offers powerful validation strategies:

• Expression system development: Utilize the evolved Mb-PylRS/Pyl-tRNA pair system that has been optimized for site-specific incorporation of ε-N-crotonyllysine (Kcr) into proteins in both E. coli and mammalian cells .

• Recombinant protein production: Generate recombinant H4 proteins with:

  • Site-specific Kcr at position 5 only

  • Kcr at alternative positions (e.g., K8, K12, K16)

  • Combinations of Kcr and other modifications

• Antibody validation: Use these defined protein standards to:

  • Assess binding affinity and specificity of the antibody

  • Determine minimum detection limits

  • Evaluate cross-reactivity with other acyl modifications

• Functional studies: Introduce these site-specifically modified histones into nucleosome reconstitution systems or cell-free transcription assays to determine the direct functional impact of K5 crotonylation.

• Western blot standards: Use defined amounts of these recombinant proteins as quantitative standards for estimating relative H4K5cr levels in biological samples .

What are common troubleshooting strategies for weak or absent signals when using Crotonyl-HIST1H4A (K5) Antibody?

When encountering signal issues with H4K5cr antibody detection:

• Sample preparation optimization:

  • Ensure complete cell lysis and histone extraction

  • Add HDAC/decrotonylase inhibitors (sodium butyrate, TSA, nicotinamide) to buffers

  • Avoid freeze-thaw cycles of extracted histones

• Antibody-specific adjustments:

  • Titrate antibody concentration (try 1:500, 1:1000, 1:2000)

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

  • Try different blocking agents (BSA vs. casein)

• Signal enhancement strategies:

  • Increase sample loading (up to 30 μg for total cell lysate)

  • Use high-sensitivity ECL substrates for Western blot

  • Try biotin-streptavidin amplification for immunostaining

• Biological considerations:

  • Verify crotonylation levels are detectable in your sample (some cell types have naturally low levels)

  • Consider treating cells with HDAC inhibitors to increase global crotonylation

  • Use metabolic interventions to boost crotonyl-CoA levels (e.g., crotonate supplementation) .

How should researchers address contradictory results between different detection methods using the same Crotonyl-HIST1H4A (K5) Antibody?

When facing inconsistent results across different techniques:

• Technique-specific considerations:

  • Western blot detects denatured proteins, while IF/IP work with native conformations

  • ChIP efficiency depends on chromatin accessibility and crosslinking efficiency

  • Different applications have distinct sensitivity thresholds

• Systematic validation approach:

  • Use positive control samples known to contain H4K5cr for all techniques

  • Compare multiple antibody sources/lots across techniques

  • Perform peptide competition assays in each technique separately

• Technical refinement:

  • Optimize fixation/extraction protocols for each technique

  • Consider epitope masking in different contexts

  • Evaluate buffer compatibility with maintaining the crotonyl modification

• Orthogonal validation:

  • Employ mass spectrometry to quantify H4K5cr levels independently

  • Use genetic approaches (site-specific incorporation of Kcr) as standards

  • Apply alternative detection methods (e.g., crotonyl-reader domain binding assays) .

What storage and handling recommendations ensure optimal performance of Crotonyl-HIST1H4A (K5) Antibody?

To maintain antibody performance over time:

• Storage conditions:

  • Store at -20°C or -80°C for long-term storage

  • Avoid repeated freeze-thaw cycles by preparing small working aliquots

  • Some formulations contain 50% glycerol allowing -20°C storage

• Working solution handling:

  • Keep on ice during experiments

  • Return to 4°C promptly after use

  • Do not leave at room temperature for extended periods

• Stability considerations:

  • Most antibody preparations remain stable for 12 months from receipt when properly stored

  • Monitor performance periodically using positive control samples

  • Record lot numbers and performance in laboratory notebooks

• Buffer compatibility:

  • Standard antibody buffer (0.01M PBS, pH 7.4 with preservatives) maintains stability

  • Sodium azide (0.02-0.05%) or Proclin-300 (0.03%) can be added as preservatives for working solutions

  • Avoid buffer additions that might affect the crotonyl modification (strong reducing agents, metal ions) .

How can Crotonyl-HIST1H4A (K5) Antibody be utilized in single-cell epigenomic technologies?

Adapting H4K5cr antibodies for single-cell applications:

• Single-cell CUT&Tag/CUT&RUN:

  • Protocol optimization for using Crotonyl-HIST1H4A (K5) Antibody in microfluidic-based single-cell platforms

  • Integration with single-cell RNA-seq to correlate H4K5cr patterns with gene expression at single-cell resolution

  • Computational approaches for handling sparse data typical of single-cell epigenomic profiles

• Mass cytometry (CyTOF) applications:

  • Metal-conjugated H4K5cr antibodies for high-parameter analysis

  • Multiplexing with other histone modification antibodies and cellular markers

  • Clustering algorithms to identify cell subpopulations with distinct H4K5cr signatures

• In situ visualization:

  • Combining H4K5cr immunofluorescence with RNA-FISH for spatial context

  • Development of proximity ligation assays (PLA) to detect H4K5cr co-occurrence with reader proteins

  • Super-resolution microscopy techniques for detailed nuclear localization of H4K5cr domains

• Technical challenges:

  • Signal amplification strategies for detecting low-abundance modifications

  • Minimizing batch effects in single-cell epigenomic data

  • Development of analytical pipelines specific for single-cell histone modification data .

What is known about the relationship between H4K5 crotonylation and other histone modifications in the broader context of the histone code?

Understanding H4K5cr in the complex landscape of histone modifications:

• Modification crosstalk patterns:

  • H4K5cr may have antagonistic relationships with H4K5 methylation

  • Potential synergistic or sequential relationships with other nearby modifications (H4K8ac, H4K12ac)

  • Undetermined relationships with modifications on other histones (H3K27ac, H3K4me3)

• Research approaches:

  • Sequential ChIP to identify co-occurrence with other modifications

  • Mass spectrometry of histone peptides to quantify combinatorial modification patterns

  • Reconstituted designer nucleosomes with specific modification combinations

• Reader protein interactions:

  • Identify proteins that specifically recognize H4K5cr versus H4K5ac

  • Determine how neighboring modifications affect reader protein binding

  • Investigate whether H4K5cr creates unique protein interaction surfaces

• Functional implications:

  • Different combinations may signal for distinct biological processes

  • Temporal dynamics during cellular transitions and development

  • Evolution of the modification pattern across species .

How might metabolic changes affect H4K5 crotonylation patterns detectable with this antibody?

The intersection of metabolism and histone crotonylation:

• Crotonyl-CoA metabolism regulation:

  • Fatty acid oxidation produces crotonyl-CoA as an intermediate

  • Manipulation of β-oxidation pathways can alter cellular crotonyl-CoA pools

  • Short-chain fatty acid supplementation (particularly crotonate) can increase histone crotonylation

• Experimental approaches:

  • Metabolic interventions (glucose deprivation, fatty acid supplementation) followed by H4K5cr ChIP-seq

  • Isotope tracing to track the incorporation of labeled precursors into histone crotonylation

  • Genetic manipulation of key metabolic enzymes (ACADS, HADH) to alter crotonyl-CoA levels

• Physiological contexts:

  • Changes in crotonylation during cellular stress responses

  • Tissue-specific patterns of crotonylation reflecting metabolic specialization

  • Altered crotonylation in metabolic diseases (obesity, diabetes, cancer)

• Technical considerations:

  • Standardized cell culture conditions to minimize metabolic variability

  • Careful timing of sample collection relative to metabolic interventions

  • Integration of metabolomic data with histone modification profiling .