Crotonyl-HIST1H2BC (K16) Antibody

What is the Crotonyl-HIST1H2BC (K16) antibody and what does it detect?

The Crotonyl-HIST1H2BC (K16) antibody is a polyclonal antibody that specifically recognizes crotonylation at lysine 16 (K16) of histone H2B type 1-C/E/F/G/I. This post-translational modification represents an important epigenetic mark distinct from the more extensively studied histone acetylation . The antibody is raised in rabbits against a peptide sequence surrounding the crotonyl-lysine (K16) site derived from histone H2B type 1-C/E/F/G/I .

How does histone crotonylation differ from histone acetylation?

While both crotonylation and acetylation are short-chain lysine acylations that occur on histone proteins, emerging evidence suggests they are functionally distinct. Crotonylation features a four-carbon chain with a double bond (CH₃-CH=CH-CO-), which differs structurally from acetylation's simpler two-carbon group (CH₃-CO-) . This structural difference affects recognition by reader proteins and binding dynamics, contributing to distinct functional outcomes. Additionally, histone crotonylation has a more pronounced ability to regulate gene expression and reflects cellular metabolic status, with competition for modification sites potentially serving as an important epigenetic regulatory mechanism .

What are the primary applications for Crotonyl-HIST1H2BC (K16) antibody in research?

The Crotonyl-HIST1H2BC (K16) antibody has been validated for multiple experimental applications with specific recommended dilutions:

These applications enable researchers to investigate the presence, abundance, and genomic distribution of crotonylated histone H2B at lysine 16 .

What is the biological significance of histone H2B crotonylation?

Histone H2B crotonylation represents an important epigenetic modification involved in multiple biological processes. Nucleosomes, which consist of DNA wrapped around histone octamers, compact DNA into chromatin and regulate DNA accessibility to cellular machinery . Histone H2B, as a core nucleosome component, plays a central role in transcription regulation, DNA repair, DNA replication, and chromosomal stability. Crotonylation at K16 of H2B contributes to the "histone code" that regulates DNA accessibility . This modification has been implicated in signal-dependent gene activation, spermatogenesis, and tissue injury responses, providing a link between cellular metabolism and epigenetic regulation .

How do enzymes regulate histone crotonylation, and what implications does this have for experimental design?

Histone crotonylation is enzymatically regulated by several histone crotonyltransferases (HCTs), including p300/CBP and members of the MYST family (MOF in humans, Esa1 in yeast) . These enzymes exhibit varying efficiencies in crotonylation activity. P300, for example, can accommodate crotonyl-CoA in its active site but performs crotonylation much less efficiently (64-fold less) than acetylation due to steric constraints .

The mechanism involves positioning of crotonyl-CoA in the substrate-binding tunnel, which requires displacement of the crotonyl group into a "back hydrophobic pocket" within the active site to enable orientation suitable for acyl-chain transfer . For experimental design, researchers should consider:

• The differential activity of various HCTs when studying specific crotonylation marks

• The competition between acylation types when manipulating cellular metabolism

• The potential requirement for additional cellular factors that enhance crotonyltransferase activity in cells compared to in vitro conditions

• The use of appropriate enzyme inhibitors or genetic modifications to isolate effects specific to crotonylation versus other acylations

What factors affect the specificity and reliability of Crotonyl-HIST1H2BC (K16) antibody detection?

Several factors can influence the specificity and reliability of Crotonyl-HIST1H2BC (K16) antibody detection:

• Cross-reactivity: As a polyclonal antibody, it may recognize similar epitopes across multiple H2B variants (H2BC4, H2BC6, H2BC7, H2BC8, H2BC10) due to sequence similarity .

• Epitope accessibility: Proper sample preparation is crucial as the crotonylated lysine residue may be obscured in certain chromatin conformations.

• Modification density: Low abundance of the modification can affect detection sensitivity, particularly in western blots and immunofluorescence.

• Buffer conditions: The antibody is stored in 50% glycerol with 0.03% Proclin 300 in 0.01M PBS (pH 7.4), which should be considered when designing experiments .

• Freezing conditions: Repeated freeze-thaw cycles should be avoided to maintain antibody integrity .

Researchers should include appropriate controls to validate specificity, including:

• Peptide competition assays using crotonylated vs. acetylated peptides

• Samples treated with decrotonylase enzymes

• Genetic knockouts of crotonyltransferases

How can Crotonyl-HIST1H2BC (K16) antibody be integrated into active learning frameworks for antibody-antigen interaction studies?

Recent research has explored active learning (AL) techniques to enhance the selection and sequencing of antigens in iterative laboratory experiments, aiming to reduce the number of experiments needed to accurately predict antibody-antigen binding . For researchers incorporating Crotonyl-HIST1H2BC (K16) antibody into such frameworks:

• Model-based strategies: Query-by-Committee (QBC) and Gradient-Based Uncertainty approaches can be applied to identify the most informative antibody-antigen pairs for labeling, potentially including interactions with crotonylated histone variants .

• Diversity-based approaches: Methods like Hamming Average Distance have shown significant performance gains (1.795% increase in area under the active learning curve compared to random selection) and can reduce required antigen mutant variants by up to 35% .

• Efficiency improvements: Implementing these AL strategies could significantly reduce the experimental iterations needed when using Crotonyl-HIST1H2BC (K16) antibody to study binding interactions with various histone variants or mutants .

• Pre-selection optimization: Clustering-based approaches like average Hamming distance can be applied before iterative learning begins, enabling more efficient selection of antigen variants to test with the antibody .

Western Blot Sample Preparation:

• Extract histones using acid extraction (0.2N HCl) or commercial histone extraction kits to ensure enrichment of histone proteins.

• Include histone deacetylase inhibitors (e.g., sodium butyrate) and protease inhibitors in all buffers to preserve modifications.

• Use SDS-PAGE with 15-18% gels for optimal histone protein separation.

• Transfer to PVDF membrane (preferred over nitrocellulose for histone proteins).

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

• Use the antibody at 1:100-1:1000 dilution as recommended .

ChIP Protocol Optimization:

• Crosslink cells with 1% formaldehyde for 10 minutes at room temperature.

• Lyse cells and sonicate chromatin to 200-500bp fragments.

• Pre-clear chromatin with protein A/G beads.

• Incubate with Crotonyl-HIST1H2BC (K16) antibody overnight at 4°C.

• Wash stringently to remove non-specific binding.

• Elute, reverse crosslinks, and purify DNA for downstream analysis.

• Include appropriate controls: input chromatin, IgG control, and positive control using antibodies against known abundant histone marks.

How can researchers distinguish between crotonylation and other acylation marks during experimental analysis?

Distinguishing between crotonylation and other acylation marks requires specific methodological approaches:

• Mass Spectrometry Validation:

  • Use high-resolution tandem mass spectrometry to definitively identify crotonylation by its characteristic mass shift (+68.0262 Da) compared to acetylation (+42.0106 Da).

  • Apply multiple fragmentation methods (CID, ETD, HCD) to increase confidence in modification identification.

• Antibody Specificity Controls:

  • Perform dot blot or peptide competition assays using synthetic peptides with different modifications (crotonylation, acetylation, butyrylation).

  • Pre-absorb antibody with acetylated peptides to confirm specificity for crotonylation.

• Enzymatic Approach:

  • Treat samples with specific decrotonylases (e.g., Sirt3) that remove crotonylation but not other modifications.

  • Compare results before and after enzymatic treatment to confirm specificity.

• Metabolic Manipulation:

  • Modify cellular levels of crotonyl-CoA versus acetyl-CoA to differentially affect modification types.

  • Measure changes in antibody binding following metabolic alterations.

What are the key considerations when designing ChIP-seq experiments using Crotonyl-HIST1H2BC (K16) antibody?

When designing ChIP-seq experiments with Crotonyl-HIST1H2BC (K16) antibody, researchers should consider:

• Antibody Validation:

  • Confirm specificity through Western blot showing single band at expected molecular weight for H2B.

  • Perform pilot ChIP-qPCR at known target loci before proceeding to sequencing.

• Chromatin Preparation:

  • Optimize sonication conditions to achieve consistent 200-500bp fragments.

  • Use spike-in controls (e.g., Drosophila chromatin with Drosophila-specific antibody) for normalization across samples.

• Sequencing Depth:

  • Aim for minimum 20 million uniquely mapped reads for histone modifications.

  • Consider deeper sequencing for modifications with potentially sparse distribution.

• Data Analysis Pipeline:

  • Use appropriate peak callers optimized for histone modifications (e.g., MACS2 with broad peak settings).

  • Perform differential binding analysis between experimental conditions.

  • Correlate crotonylation patterns with gene expression data and other histone marks.

• Biological Interpretation:

  • Compare crotonylation patterns with acetylation patterns to identify unique versus shared genomic regions.

  • Analyze crotonylation in the context of metabolic state and cellular function.

How can researchers integrate multi-omics approaches when studying histone crotonylation?

Integrating multi-omics approaches can provide comprehensive insights into histone crotonylation function:

• ChIP-seq + RNA-seq Integration:

  • Correlate H2BK16cr distribution with transcriptional changes.

  • Identify genes specifically regulated by crotonylation versus acetylation.

  • Use established statistical frameworks to associate chromatin states with expression patterns.

• Metabolomics Integration:

  • Measure cellular crotonyl-CoA levels alongside crotonylation patterns.

  • Correlate metabolic shifts with changes in crotonylation distribution.

  • Identify metabolic pathways that influence crotonylation levels.

• Proteomics Approaches:

  • Use proteome-wide approaches to identify all crotonylated proteins beyond histones.

  • Apply proximity labeling techniques to identify proteins interacting with crotonylated histones.

  • Quantify changes in reader protein binding following alterations in crotonylation.

• Functional Genomics:

  • Apply CRISPR-based approaches to modify enzymes regulating crotonylation.

  • Use targeted epigenome editing to manipulate crotonylation at specific loci.

  • Correlate phenotypic changes with alterations in crotonylation patterns.

What are common pitfalls when using Crotonyl-HIST1H2BC (K16) antibody and how can they be addressed?

Researchers should specifically note that the Crotonyl-HIST1H2BC (K16) antibody should be stored at -20°C or -80°C, and repeated freeze-thaw cycles should be avoided to maintain integrity .

How can researchers validate that they are specifically detecting crotonylation at K16 of HIST1H2BC?

To confirm specific detection of K16 crotonylation on HIST1H2BC:

• Peptide Competition Assays:

  • Compare binding inhibition using:

    • K16-crotonylated H2B peptide (should block binding)

    • Unmodified H2B peptide (should not block)

    • K16-acetylated H2B peptide (should show minimal blocking if antibody is specific)

    • Crotonylated peptides at other lysine positions (should show minimal blocking)

• Mutational Analysis:

  • Express wild-type H2B and K16R mutant (prevents modification)

  • Compare antibody reactivity between variants

• Mass Spectrometry Validation:

  • Immunoprecipitate with the antibody

  • Analyze by MS to confirm K16 crotonylation in the bound fraction

• Enzyme Treatment Controls:

  • Treat samples with HDAC2 (removes acetylation but not crotonylation)

  • Treat with Sirt3 (can remove crotonylation)

  • Compare antibody reactivity after these treatments

How might Crotonyl-HIST1H2BC (K16) antibody be used to explore the relationship between cellular metabolism and epigenetic regulation?

The Crotonyl-HIST1H2BC (K16) antibody offers significant potential for investigating metabolic-epigenetic connections:

• Metabolic Stress Responses:

  • Map changes in H2BK16cr distribution following alterations in cellular energy status.

  • Compare crotonylation patterns in normal versus cancer cells with dysregulated metabolism.

  • Identify metabolic pathways specifically affecting crotonylation versus other acylations.

• Nutritional Influences:

  • Track H2BK16cr changes in response to different dietary components that affect crotonyl-CoA levels.

  • Investigate fasting/feeding cycles and their impact on histone crotonylation.

  • Compare tissue-specific crotonylation patterns in different nutritional states.

• Metabolic Disease Models:

  • Examine H2BK16cr in diabetes, obesity, and other metabolic disorders.

  • Correlate changes with altered gene expression and cellular functions.

  • Investigate potential therapeutic approaches targeting crotonylation pathways.

This research direction aligns with the emerging understanding that histone crotonylation represents a mechanism by which cellular metabolic status influences epigenetic regulation .

What novel active learning approaches might improve antibody-antigen interaction predictions for crotonylated histone variants?

Building on recent advances in active learning (AL) for antibody-antigen interaction studies , several novel approaches could enhance research with crotonylated histone variants:

• Hybrid AL Strategies:

  • Combine model-based and diversity-based approaches to optimize selection criteria.

  • Implement ensemble methods that leverage multiple uncertainty measures simultaneously.

  • Develop adaptive strategies that switch between selection criteria based on learning progress.

• Transfer Learning Applications:

  • Utilize knowledge from well-characterized histone modifications to inform predictions about crotonylation.

  • Apply pre-trained models from acetylation studies with fine-tuning for crotonylation specifics.

• Multi-objective Optimization:

  • Balance exploration of diverse crotonylated variants with exploitation of promising interaction regions.

  • Incorporate biological knowledge (e.g., structural information) into selection criteria.

• Reinforcement Learning Frameworks:

  • Develop systems that learn optimal experiment selection strategies through iterative feedback.

  • Reward functions could incorporate both prediction accuracy and experimental cost/feasibility.

These approaches could reduce experimental iterations by up to 35% compared to random selection, as demonstrated by similar strategies in other antibody-antigen studies .