Crotonyl-HIST1H3A (K9) Antibody

What applications are suitable for Crotonyl-HIST1H3A (K9) antibody?

The Crotonyl-HIST1H3A (K9) antibody has been validated for multiple experimental applications including Western blot (WB), dot blot, ELISA, immunocytochemistry (ICC), immunoprecipitation (IP), and chromatin immunoprecipitation (ChIP) . When designing experiments, researchers should consider that antibody concentrations vary by application: WB (1:100-1:1000), ICC (1:20-1:200), and IP (1:200-1:2000) . For optimal results in ChIP-seq applications, the antibody has been successfully used to map genome-wide distribution of H3K9 crotonylation, particularly at transcriptional start sites (TSSs) and termination sites (TTSs) .

How should I validate the specificity of H3K9cr antibodies?

Validation of H3K9cr antibody specificity is critical for reliable results. Implement peptide dot blot assays using synthetic peptides with defined modifications to confirm target specificity . Be aware that some H3K9cr antibodies, while specific to crotonylation, may show cross-reactivity with butyrylation at the same residue . For instance, the antibody in search result shows specific reactivity to H3K9cr but cross-reacts with H3K9 butyrylation. Consider including both positive controls (synthetic H3K9cr peptides) and negative controls (unmodified H3 and other acylations) in validation assays. Additionally, mass spectrometric analysis of isolated histones provides complementary validation of H3K9cr presence in your samples .

What is the relationship between H3K9 crotonylation and gene expression?

H3K9 crotonylation positively correlates with gene expression levels. ChIP-seq analysis has revealed that genes with the highest expression quartiles display the highest occupancy of H3K9cr at their transcriptional start sites . This modification is particularly enriched during the transition from high oxygen consumption (HOC) to low oxygen consumption (LOC) phases in yeast metabolic cycles, coinciding with β-oxidation . The relative ratio of H3K9 crotonylation to acetylation is highest on periodically expressed genes involved in ribosome biogenesis and translation, suggesting a role in the precise regulation of energy-demanding, highly expressed genes . When analyzing your ChIP-seq data, correlate H3K9cr enrichment patterns with RNA-seq data to establish functional relationships with transcriptional activity in your specific biological context.

How should I design Western blot experiments to detect H3K9 crotonylation?

For optimal Western blot detection of H3K9 crotonylation, follow this methodological approach: 1) Extract histones using acid extraction to preserve post-translational modifications; 2) Load 10-20 μg of histone extracts per lane on a 15% SDS-PAGE gel to achieve good separation of histone proteins; 3) Use 5 μg/ml of anti-H3K9cr antibody (e.g., RM339 clone) ; 4) Include appropriate controls - anti-Histone H3 antibody as a loading control and anti-G6PDH as a non-histone control; 5) Consider using a crotonylation-inducing metabolite to increase H3K9cr signal, particularly in systems with low baseline crotonylation . When interpreting results, the predicted band size for H3K9cr should be approximately 15 kDa . Wash blots with TBS-T (tris-borate-sodium-0.05% Tween-20) and develop using enhanced chemiluminescence (ECL) for optimal signal detection .

What methodology should I use for ChIP-seq with H3K9cr antibodies?

For successful ChIP-seq using H3K9cr antibodies, implement this protocol: 1) Cross-link chromatin with 1% formaldehyde; 2) Sonicate to generate fragments of 200-500 bp; 3) Immunoprecipitate using anti-H3K9cr antibody; 4) Include parallel H3K9ac ChIP-seq for comparative analysis; 5) Sequence at sufficient depth (>20 million reads) to capture genome-wide distribution . During data analysis, focus on enrichment at TSSs and TTSs of RNAPII-regulated genes, as these are primary sites of H3K9cr localization . For biological interpretation, analyze the correlation between H3K9cr enrichment and gene expression levels by quartile distribution, and calculate the relative ratio of H3K9 crotonylation to acetylation at specific genomic regions to identify functionally significant patterns .

How can I quantitatively compare histone crotonylation and acetylation levels?

To quantitatively compare histone crotonylation and acetylation levels, employ a multi-faceted approach: 1) Perform parallel ChIP-seq experiments using validated antibodies specific to H3K9cr and H3K9ac; 2) Calculate the ratio of crotonylation to acetylation at specific genomic regions by normalizing read counts; 3) Correlate modification levels with gene expression data to determine functional relationships . For biochemical quantification, use in vitro modified histones as standards for Western blot or dot blot analysis. When comparing enzyme kinetics of deacetylation versus decrotonylation, prepare fully modified histones, perform reactions with defined enzyme concentrations (e.g., 0.03 μM HDAC1) and substrate concentrations (1.41 to 0.19 μM modified histone H3), and quantify reaction products at multiple time points to determine Km, Vmax, and Kcat values .

How do metabolic states influence H3K9 crotonylation patterns?

Metabolic states significantly impact H3K9 crotonylation patterns through the regulation of crotonyl-CoA availability. In yeast metabolic cycle (YMC) studies, H3K9 crotonylation exhibits dynamic changes that peak during the transition from high oxygen consumption (HOC) to low oxygen consumption (LOC) phases, coinciding with β-oxidation . This temporal pattern differs from H3K9 acetylation, which peaks during the HOC phase when acetyl-CoA levels are abundant . To investigate metabolic influences on H3K9cr in your research: 1) Manipulate cellular metabolism through nutrient limitation (e.g., glucose-limited continuous culture); 2) Monitor crotonylation dynamics across metabolic cycles; 3) Compare with asynchronous cultures in nutrient-rich media, which typically show relatively low levels of H3K9 crotonylation ; 4) Consider using short-chain fatty acids to modulate histone crotonylation levels . Notably, microbiota-derived short-chain fatty acids promote histone crotonylation, connecting chromatin modifications to gut microbiota, partially through HDAC inhibition .

How do histone deacetylases (HDACs) interact with crotonylated histones?

HDACs interact with crotonylated histones as decrotonylases, though with different kinetics than their deacetylation activity. To characterize HDAC activity on crotonylated histones: 1) Prepare in vitro crotonylated and acetylated histones; 2) Perform enzyme kinetic analysis using purified HDACs (e.g., recombinant human HDAC1); 3) Measure decrotonylation/deacetylation rates at varying substrate concentrations; 4) Calculate and compare Km, Vmax, and Kcat values for both modifications . Experimental data reveals differences in enzyme kinetics between decrotonylation and deacetylation reactions. When designing HDAC inhibition studies, consider that inhibitors may differentially affect deacetylase versus decrotonylase activities. Additionally, investigate the role of specific HDACs (HDAC1, HDAC2, HDAC3) using siRNA knockdown or selective inhibitors to determine their relative contributions to cellular H3K9cr levels .

What controls should I include in H3K9cr detection experiments?

Comprehensive controls are essential for reliable H3K9cr detection. Include these controls in your experimental design: 1) Positive controls: synthetic H3K9cr peptides or recombinant fragments with verified crotonylation; 2) Negative controls: unmodified H3 peptides and peptides with other acylations (e.g., acetylation, butyrylation); 3) Loading controls: total histone H3 antibody to normalize for histone content; 4) Technical controls: isotype-matched IgG for ChIP background assessment; 5) Biological controls: samples with artificially induced or depleted crotonylation . For Western blots, include anti-G6PDH as a non-histone control . In ChIP-seq experiments, perform parallel H3K9ac ChIP as a comparative control . When using metabolic manipulations to alter crotonylation, include time course samples to capture dynamic changes. Additionally, consider using crotonylation-inducing metabolites to increase signal-to-noise ratio in systems with low baseline crotonylation .

How do I optimize ChIP conditions for H3K9cr antibodies?

Optimizing ChIP conditions for H3K9cr antibodies requires systematic adjustment of experimental parameters. Follow this methodological approach: 1) Antibody titration: Test multiple concentrations (1:200-1:2000 dilution range) to determine optimal antibody-to-chromatin ratio ; 2) Crosslinking optimization: Compare different formaldehyde concentrations (0.5-1.5%) and incubation times (5-15 minutes) to preserve the crotonyl modification while achieving sufficient crosslinking; 3) Sonication parameters: Adjust power settings and cycle numbers to consistently generate 200-500 bp fragments without damaging epitopes; 4) Washing stringency: Test different salt concentrations in wash buffers to minimize background while maintaining specific binding . Evaluate optimization results through qPCR of known H3K9cr-enriched regions before proceeding to sequencing. For challenging samples with low crotonylation levels, consider using carriers (e.g., glycogen or tRNA) during immunoprecipitation to improve recovery. Document all optimization steps methodically to establish a reproducible protocol for your specific experimental system.

What is the relationship between H3K9 crotonylation and cellular metabolism?

H3K9 crotonylation serves as a chromatin-based sensor of cellular metabolic state, linking epigenetic regulation to metabolic fluctuations. This relationship manifests through: 1) Dynamic changes in H3K9cr levels across metabolic cycles, peaking during the HOC to LOC transition when β-oxidation occurs ; 2) Temporal segregation from H3K9 acetylation, which peaks during the HOC phase when acetyl-CoA levels are high ; 3) Connection to microbiota-derived short-chain fatty acids, which can promote histone crotonylation . To investigate this relationship, monitor crotonylation levels under various metabolic conditions: glucose limitation, fatty acid oxidation inhibition/stimulation, or modulation of crotonyl-CoA production. Notably, asynchronous yeast cultures in nutrient-rich media show relatively low H3K9cr levels compared to metabolically synchronized cultures, highlighting the utility of controlled metabolic systems for studying this modification . The correlation between H3K9cr and metabolic genes suggests a potential feedback mechanism where metabolic state influences chromatin structure, which in turn regulates metabolic gene expression.

How do Taf14 and other reader proteins recognize H3K9 crotonylation?

Reader proteins like Taf14 specifically recognize H3K9 crotonylation to mediate its functional outcomes. The recognition mechanism involves: 1) The YEATS domain of Taf14, which forms a binding pocket that accommodates the crotonyl modification; 2) Specific interactions that distinguish crotonylation from other acylations like acetylation . To study reader protein interactions: 1) Perform in vitro binding assays with recombinant YEATS domains and modified histone peptides; 2) Measure binding affinities (Kd values) for different acyl modifications; 3) Use structural approaches (X-ray crystallography, NMR) to characterize the molecular basis of selectivity; 4) Conduct cellular studies with YEATS domain mutants to assess functional consequences of disrupted crotonylation recognition. The link between Taf14 recognition of H3K9cr and metabolic state suggests that reader proteins may function as metabolic sensors, translating changes in cellular metabolism to altered gene expression programs through selective binding to crotonylated chromatin .

What is the functional significance of the temporal dynamics between H3K9 crotonylation and acetylation?

The temporal dynamics between H3K9 crotonylation and acetylation reflect a sophisticated regulatory mechanism coordinating gene expression with metabolic cycles. Functionally, this temporal segregation serves to: 1) Fine-tune the expression of energy-demanding genes, with acetylation promoting initial activation and crotonylation potentially extending or modifying the transcriptional response ; 2) Establish a crotonylation-to-acetylation ratio that marks specific gene classes, particularly those involved in ribosome biogenesis and translation ; 3) Create distinct chromatin states that respond to changing metabolic conditions. To investigate these dynamics: 1) Perform time-course ChIP-seq for both modifications during metabolic transitions; 2) Correlate modification patterns with nascent transcription data (e.g., NET-seq or PRO-seq); 3) Experimentally manipulate the ratio through HDAC inhibition or metabolite supplementation; 4) Assess the consequences on gene expression timing and amplitude . Notably, genes with the highest amplitude of periodic expression in metabolic cycles show high H3K9cr/H3K9ac ratios during the HOC to LOC transition, suggesting that this ratio is a key regulatory feature rather than just a consequence of changing metabolites .

How can I combine H3K9cr ChIP-seq with other genomic approaches for comprehensive epigenetic analysis?

Integrating H3K9cr ChIP-seq with complementary genomic approaches enables comprehensive epigenetic analysis. Implement this methodological framework: 1) Parallel ChIP-seq for multiple histone modifications (H3K9ac, H3K4me3, H3K27ac) to create comprehensive epigenetic maps; 2) ATAC-seq or DNase-seq to correlate H3K9cr with chromatin accessibility; 3) RNA-seq or NET-seq to link modification patterns with transcriptional output; 4) Hi-C or ChIA-PET to examine three-dimensional chromatin organization in relation to H3K9cr enrichment . For data integration, use computational approaches that identify statistically significant correlations between datasets and employ visualization tools that display multiple data tracks aligned to genomic coordinates. Consider time-course experiments that capture the dynamics of H3K9cr in relation to other epigenetic features during biological processes such as cell differentiation or metabolic adaptation. This integrated approach will reveal how H3K9cr functions within the broader epigenetic landscape to regulate gene expression and chromatin structure.