Crotonyl-HIST1H2AG (K125) Antibody

Basic Research Questions

• What is the molecular target of Crotonyl-HIST1H2AG (K125) Antibody?

  Crotonyl-HIST1H2AG (K125) Antibody specifically recognizes histone H2A that has been post-translationally modified by crotonylation at lysine 125. This antibody targets the human histone H2A protein (UniProt ID: P0C0S8) . The antibody is developed using a synthetic peptide sequence surrounding the crotonylated lysine 125 site derived from Human Histone H2A type 1 . This target has several synonyms in the literature, including H2AC11, H2AFP, H2AC13, H2AFC, and others . As a core component of nucleosomes, H2A plays critical roles in chromatin structure, DNA accessibility regulation, transcription, DNA repair, and replication .

• What applications has this antibody been validated for?

  According to multiple sources, Crotonyl-HIST1H2AG (K125) Antibody has been validated for the following applications:

  Application	Validated	Recommended Dilution
  ELISA	Yes	Lot specific
  IF/ICC	Yes	1:50-1:200
  ChIP	Yes	Lot specific
  Western Blot	Yes (for some versions)	1:1000
  Peptide Array	Yes (for some versions)	Lot specific

  The specific applications may vary slightly between manufacturers, and optimal dilutions should be determined experimentally for each specific application and sample type .

• What are the critical handling and storage parameters for this antibody?

  For optimal performance, Crotonyl-HIST1H2AG (K125) Antibody requires specific handling and storage conditions:

  • Storage temperature: Upon receipt, store at -20°C or -80°C

  • Avoid repeated freeze/thaw cycles as this can diminish antibody performance

  • The antibody is typically supplied in a stabilizing buffer containing:

    • Preservative: 0.03% Proclin 300

    • Constituents: 50% Glycerol, 0.01M PBS, pH 7.4

  • The antibody should be aliquoted upon receipt to minimize freeze/thaw cycles

  • When working with the antibody, maintain cold chain practices and follow manufacturer-specific recommendations for each experimental application

Intermediate Research Questions

• How does histone H2A crotonylation influence chromatin biology and gene regulation?

  Histone crotonylation represents a novel histone post-translational modification with significant biological implications:

  • Histone lysine crotonylation (Kcr) functions as a robust indicator of active promoters

  • Particularly important in male germ cell differentiation as a regulatory signal

  • Crotonylation impacts DNA accessibility by altering chromatin structure, thereby influencing various DNA-dependent processes

  • H2A lysine crotonylation participates in a complex "histone code" alongside other modifications to regulate gene expression

  • Crotonylation at specific sites like H2AK119 has been shown to exist simultaneously with ubiquitination, with these modifications being reversibly regulated under conditions such as replication stress

• What is the relationship between crotonylation and ubiquitination at H2AK119?

  Research has revealed an intricate interplay between crotonylation and ubiquitination at H2AK119:

  • H2AK119cr (crotonylation) and H2AK119ub (ubiquitination) coexist in cells and are dynamically regulated in response to replication stress

  • SIRT1 deacetylase mediates H2AK119 crotonylation, which serves as a prerequisite for subsequent BMI1-mediated ubiquitination at this site

  • Under replication stress conditions, SIRT1 removes H2AK119cr, allowing for BMI1-mediated H2AK119ub

  • The enrichment of H2AK119ub at reversed replication forks promotes RNA Pol II removal, suppresses transcription near stalled replication forks, and reduces R-loop and double-strand break formation

  • This dynamic switching mechanism is crucial for resolving transcription-replication conflicts during cellular stress

• How can I confirm the specificity of Crotonyl-HIST1H2AG (K125) Antibody?

  Several approaches can validate antibody specificity:

  • Peptide array analysis: Test antibody against multiple modified and unmodified histone peptides to evaluate cross-reactivity

  • Western blot analysis with appropriate controls: Compare signal from samples with known high and low levels of the modification

  • Competitive binding assays with the immunizing peptide: Signal should decrease with increasing amounts of competing peptide

  • Testing in knockout models: Use of SIRT1-KO cells, which would be expected to show increased crotonylation due to lack of decrotonylase activity

  • Isotope labeling experiments: Culture cells with D4-crotonate (labeled crotonate) and confirm antibody detection of the labeled modification by mass spectrometry

Advanced Research Questions

• What methodologies can be used to study dynamic changes in histone crotonylation?

  To investigate the dynamics of histone crotonylation:

  • In vitro de-crotonylation assays: Mix purified histone substrate with recombinant SIRT1 enzyme in reaction buffer (50 mM Tris, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 3mM NAD+/[Zn2+], 1 mM MgCl2, and 1 mM DTT) at 37°C, followed by western blot analysis

  • Metabolic labeling with crotonate: Culture cells with crotonate or D4-crotonate to increase cellular crotonylation or track newly added crotonyl groups

  • ChIP-seq time-course experiments: Map genome-wide distribution of crotonylated histones under different conditions or time points

  • Integrated MS approaches: Combine multiple proteolytic methods as described in the literature for comprehensive PTM mapping:

    1. Tryptic digestion without chemical derivatization

    2. Tryptic peptides with post-digestion propionylation

    3. Tryptic peptides from propionylated histones

    4. In-gel digestion of individual histone proteins

• How can I optimize ChIP protocols using Crotonyl-HIST1H2AG (K125) Antibody?

  For successful ChIP experiments with this antibody:

  • Sample preparation: Ensure proper crosslinking and chromatin shearing to appropriate fragment size (typically 200-500bp)

  • Antibody concentration: Titrate antibody amounts; typically start with manufacturer recommendations and optimize

  • Pre-clearing step: Include to reduce background from non-specific binding

  • Controls: Always include:

    • Input control (pre-immunoprecipitation chromatin)

    • IgG control (same species as primary antibody)

    • Positive control (known region enriched for your target)

  • Washing conditions: Optimize stringency of wash buffers to balance between signal retention and background reduction

  • For peptide immunoprecipitation after tryptic digestion: Consider the approach described for D4-crotonate labeled samples, followed by HPLC/MS/MS analysis

• How does environmental stress affect crotonylation patterns, and what techniques can detect these changes?

  Research indicates that cellular stress impacts histone crotonylation:

  • Replication stress causes dynamic switching between H2AK119cr and H2AK119ub, mediated by SIRT1 and BMI1

  • To study these changes:

    • Use synchronized cell populations to control for cell cycle effects

    • Induce stress with appropriate agents (e.g., hydroxyurea for replication stress)

    • Perform time-course experiments to track modification dynamics

    • Use ChIP-seq to map genome-wide redistribution of crotonylation

    • Combine with transcriptome analysis to correlate with gene expression changes

    • Consider metabolomic analysis to measure cellular crotonyl-CoA levels, which function as crotonylation donors

  • Validation should include multiple techniques (western blot, immunofluorescence, mass spectrometry)

• What analytical framework can be used to investigate synergistic effects involving histone crotonylation?

  To study how crotonylation interacts with other histone modifications or genetic factors:

  • Design combinatorial perturbation experiments as described in the literature for studying gene-gene, gene-environment interactions, or genotype-specific drug responses

  • Key experimental design considerations:

    • Include all necessary controls (individual perturbations and combinations)

    • Calculate appropriate sample sizes using power analysis functions to detect differences between conditions

    • Minimize batch effects and technical variation

    • For RNA-seq analysis, ensure raw read counts are available

  • Analytical approach:

    • Query interactions between two or more perturbagens

    • Resolve the extent of non-additive (synergistic) interactions

    • Compare observed combined effects against predicted additive effects from individual perturbations

  • Validate findings with:

    • Functional assays relevant to the biological process being studied

    • Protein-level confirmation of transcriptional changes

    • Assessment of cellular phenotypes resulting from modification changes

• What methodological advances have enhanced detection and characterization of histone crotonylation?

  Recent technical developments have improved crotonylation analysis:

  • Integrated mass spectrometry approaches combining multiple proteolytic strategies to achieve 87-100% sequence coverage of histones

  • OFFGEL isoelectric focusing for efficient peptide separation prior to MS analysis

  • High-sensitivity MS instruments like LTQ Orbitrap Velos for comprehensive PTM identification

  • Development of specific antibodies recognizing crotonylated residues, enabling:

    • Western blot detection

    • Immunofluorescence imaging

    • ChIP applications

    • Peptide immunoprecipitation

  • Metabolic labeling with isotope-tagged crotonate (D4-crotonate) to track newly added modifications

  • Peptide arrays for evaluating antibody specificity and cross-reactivity with other modifications

• How can computational approaches enhance analysis of crotonylation datasets?

  Computational methods to analyze histone crotonylation data include:

  • Analysis framework for combinatorial perturbation experiments that can:

    • Identify synergistic vs. additive effects of multiple factors

    • Quantify the extent of non-additive interactions

    • Calculate statistical power requirements for experiment design

  • ChIP-seq analysis pipelines to:

    • Map distribution of crotonylation across the genome

    • Correlate with other genomic features (promoters, enhancers, etc.)

    • Integrate with transcriptome data to link modification to gene expression

  • Mass spectrometry data analysis workflows for:

    • Identification of crotonylated peptides from complex mixtures

    • Quantification of modification abundance

    • Comparison across different conditions

  • Consideration of statistical power: Functions to calculate needed sample sizes based on empirical measurement of variance to be adequately powered for detecting differences between conditions