Crotonyl-HIST1H4A (K16) Antibody

What is histone H4K16 crotonylation and what is its biological significance?

Histone H4K16 crotonylation is a post-translational modification that occurs at lysine 16 of histone H4. Like acetylation, crotonylation is associated with transcriptionally active chromatin regions. Crotonylation at H4K16 contributes to chromatin decondensation by disrupting histone-DNA interactions, which subsequently affects DNA accessibility to cellular machinery involved in transcription, DNA repair, and replication. This modification appears to work in concert with other histone marks to regulate gene expression and genome maintenance .

How do crotonylation and acetylation at H4K16 differ functionally?

While both modifications neutralize the positive charge of lysine residues, crotonylation involves the addition of a four-carbon crotonyl group, which is bulkier than the two-carbon acetyl group. This structural difference results in distinct functional outcomes:

• Binding partners: Crotonylation and acetylation are recognized by different reader proteins

• Stability: Crotonyl marks are generally more resistant to deacylation than acetyl marks

• Gene regulation: H4K16 crotonylation appears in specific genomic contexts that may differ from those of H4K16 acetylation

• Cellular metabolism: Crotonylation is more closely linked to cellular metabolic state through crotonyl-CoA levels

H4K16 acetylation is known to be associated with euchromatin formation, transcriptional regulation, DNA damage repair, and cell senescence, while crotonylation may have overlapping but distinct functions .

What are the recommended applications for Crotonyl-HIST1H4A (K16) Antibody?

Based on validation data, the Crotonyl-HIST1H4A (K16) Polyclonal Antibody has been confirmed for the following applications:

The antibody specifically recognizes the crotonylation at lysine 16 of human histone H4 (accession number P62805) and shows minimal cross-reactivity with other histone modifications .

What controls should be included when using Crotonyl-HIST1H4A (K16) Antibody?

For rigorous experimental design, researchers should include the following controls:

• Positive control: Cell lines or tissues known to exhibit H4K16 crotonylation (e.g., actively dividing cells)

• Negative control: Samples treated with histone decrotonylase enzymes

• Peptide competition assay: Pre-incubation of antibody with crotonylated and non-crotonylated peptides

• Isotype control: Non-specific rabbit IgG to assess background binding

• Cross-reactivity control: Recombinant histone H4 with various modifications (acetylation, methylation) to ensure specificity

These controls help validate antibody specificity and enhance the reliability of experimental results.

How to optimize ChIP protocols specifically for Crotonyl-HIST1H4A (K16) Antibody?

For optimal ChIP results using Crotonyl-HIST1H4A (K16) Antibody, consider the following modifications to standard protocols:

• Crosslinking: Use dual crosslinking with 1.5 mM EGS (ethylene glycol bis[succinimidylsuccinate]) for 30 minutes followed by 1% formaldehyde for 10 minutes to better preserve crotonylation marks

• Sonication conditions: 10-12 cycles (30 seconds ON/OFF) to generate fragments of 200-500 bp

• Antibody incubation: 4-8 μg of chromatin DNA with antibody pre-bound to magnetic beads at 4°C overnight with rotation

• Washing conditions: Two washes with ChIP buffer followed by two washes with TE buffer

• Elution: TE containing 1% SDS at 65°C overnight with proteinase K

This optimized protocol has been adapted from successful ChIP procedures for other histone modifications .

What are the best approaches to validate the specificity of Crotonyl-HIST1H4A (K16) Antibody?

Comprehensive validation of Crotonyl-HIST1H4A (K16) Antibody should include:

• Peptide array analysis: Testing antibody against a panel of modified and unmodified histone peptides

• Dot blot analysis: Using synthetic peptides with various modifications at K16 and surrounding residues

• Western blot with mutation constructs: Expressing K16A mutants of histone H4 to confirm specificity

• Mass spectrometry validation: Confirming the identity of immunoprecipitated proteins

• Knockout/knockdown validation: Using CRISPR to delete histone variants or enzymes responsible for crotonylation

• Competition assays: Pre-incubating antibody with crotonylated and non-crotonylated peptides before immunostaining

This multi-method approach ensures high confidence in antibody specificity, critical for interpreting experimental results.

How to interpret overlapping signals between crotonylation and other histone modifications?

When analyzing potentially overlapping signals:

• Perform sequential ChIP (re-ChIP) to determine co-occurrence of modifications

• Use high-resolution microscopy with spectral unmixing for co-localization studies

• Compare ChIP-seq datasets with publicly available datasets for other modifications

• Employ bioinformatic tools to identify statistically significant overlaps

• Validate functional relationships using genetic or chemical perturbation of specific modification pathways

Remember that H4K16 can be modified by various acylations, including acetylation, which is known to be associated with transcriptional activation and chromatin decondensation .

What might cause inconsistent results when using Crotonyl-HIST1H4A (K16) Antibody?

Common causes of inconsistency include:

• Sample preparation variability: Inconsistent fixation or extraction methods

• Cellular metabolic state: Variations in crotonyl-CoA levels affecting global crotonylation

• Technical factors:

  • Antibody storage conditions (avoid repeated freeze-thaw cycles)

  • Incubation time and temperature variations

  • Buffer composition differences

• Biological factors:

  • Cell cycle stage (crotonylation patterns change during cell cycle)

  • Culture conditions affecting metabolic state

  • Cell density variations

To minimize inconsistency, standardize protocols and maintain detailed records of experimental conditions .

How to address potential cross-reactivity with other histone modifications?

To address cross-reactivity concerns:

• Perform ELISA testing against a panel of modified peptides (acetylation, methylation, other acylations)

• Include appropriate blocking agents (5% BSA or 5% non-fat milk) in antibody diluent

• Pre-absorb antibody with potential cross-reactive peptides

• Validate specificity using recombinant histones with defined modifications

• Compare results with alternative antibody clones from different vendors

• Include lysine-to-alanine substitution controls in overexpression studies

This systematic approach helps distinguish genuine signals from potential artifacts due to cross-reactivity .

What are the recommended approaches for quantifying H4K16 crotonylation levels?

For accurate quantification of H4K16 crotonylation:

For all methods, include appropriate controls and reference standards for accurate quantification .

What are the optimal buffer conditions for Crotonyl-HIST1H4A (K16) Antibody in different applications?

Buffer optimization is critical for antibody performance across applications:

When working with crotonylation antibodies, including HDAC inhibitors (e.g., sodium butyrate) in buffers can help preserve the modification during extraction and processing .

How to establish the relationship between H4K16 crotonylation and gene expression?

To correlate H4K16 crotonylation with gene expression:

• Perform ChIP-seq for H4K16cr and RNA-seq on matching samples

• Use bioinformatic tools to correlate crotonylation peaks with transcript levels

• Generate heatmaps showing distribution of crotonylation relative to transcription start sites

• Perform perturbation experiments:

  • Modulate cellular crotonyl-CoA levels

  • Inhibit or overexpress enzymes responsible for crotonylation/decrotonylation

• Validate findings using reporter assays for specific target genes

Analysis typically reveals that H4K16 crotonylation, like acetylation, is enriched around transcription start sites of active genes .

What techniques can be combined with Crotonyl-HIST1H4A (K16) Antibody for multimodal analysis?

Integrative approaches include:

• ChIP-seq followed by RNA-seq to correlate chromatin state with transcriptional output

• CUT&RUN or CUT&Tag for higher resolution mapping of crotonylation sites

• Co-immunoprecipitation to identify reader proteins recognizing H4K16cr

• Mass spectrometry for comprehensive histone modification profiling

• Live-cell imaging with fluorescently labeled antibody fragments

• Proximity ligation assay (PLA) to detect co-occurrence with other modifications

These combinatorial approaches provide deeper insights into the functional significance of H4K16 crotonylation in different biological contexts .

How does H4K16 crotonylation change during cellular differentiation and development?

Understanding developmental dynamics of H4K16 crotonylation requires:

• Temporal profiling across differentiation stages

• Tissue-specific mapping in developing organisms

• Correlation with metabolic changes that affect crotonyl-CoA levels

• Comparison with other histone modifications during development

• Analysis of writer/eraser enzyme expression patterns during differentiation

Current research suggests that histone crotonylation, like acetylation, undergoes significant remodeling during cellular differentiation and development, often correlating with changes in gene expression programs .

How to analyze ChIP-seq data specifically for histone crotonylation marks?

For robust ChIP-seq analysis of H4K16 crotonylation:

• Quality control: Assess sequencing depth (>20 million mapped reads recommended)

• Peak calling: Use MACS2 with broad peak settings for histone modifications

• Normalization: Apply input control and spike-in normalization for quantitative comparisons

• Visualization: Generate heatmaps centered on transcription start sites or enhancers

• Comparative analysis: Overlay with other histone modifications and transcription factor binding sites

• Motif analysis: Identify DNA sequences enriched in crotonylated regions

• Pathway analysis: Associate crotonylation patterns with biological functions

As with acetylation marks, H4K16 crotonylation is typically enriched around transcription start sites and correlates with gene activity .

What are the recommended protocols for sequential ChIP to study co-occurrence with other modifications?

For effective sequential ChIP (re-ChIP):

• First immunoprecipitation: Use standard ChIP protocol with Crotonyl-HIST1H4A (K16) Antibody

• Elution: Use 10 mM DTT at 37°C for 30 minutes (preserves modifications)

• Dilution: Dilute eluted chromatin 1:10 in ChIP buffer before second IP

• Second immunoprecipitation: Use antibody against the second modification of interest

• Final elution: Use standard elution buffer with 1% SDS

• Controls: Include single ChIP and reverse order re-ChIP as controls

This approach allows determination of whether different histone modifications co-occur on the same nucleosomes, providing insights into their functional relationships .