Crotonyl-HIST1H2BC (K12) Antibody

What is histone crotonylation and why is the Crotonyl-HIST1H2BC (K12) modification significant?

Histone crotonylation is a post-translational modification recently discovered in histones that plays a crucial role in regulating gene expression and chromatin architecture. Specifically, crotonylation of Histone H2BC at lysine 12 (K12) represents a specific modification site with biological significance. This modification contributes to chromatin remodeling processes that influence DNA accessibility to transcriptional machinery. The significance of this modification lies in its distinct role from other histone modifications like acetylation and methylation, potentially serving as a unique regulatory mechanism in gene expression. Crotonylation at K12 is particularly important as a marker for active gene transcription in multiple cell types and developmental contexts. Understanding this modification provides insights into epigenetic regulation mechanisms that govern cellular processes including development, differentiation, and pathological conditions .

How does crotonylation at K12 differ from acetylation at the same position?

Crotonylation and acetylation at the K12 position of Histone H2BC represent distinct post-translational modifications with different molecular structures and potentially different biological functions. Structurally, crotonylation involves the addition of a crotonyl group (C4H5O) to the lysine residue, which contains an extended four-carbon chain with a double bond, making it bulkier than the acetyl group (C2H3O) added during acetylation. This structural difference appears to result in distinct downstream effects on chromatin structure and protein interactions. While both modifications neutralize the positive charge of lysine and generally promote a more open chromatin state, research indicates that crotonylation may have stronger effects on transcriptional activation compared to acetylation. The regulatory enzymes ("writers" and "erasers") that control these modifications also differ, with specific crotonylation regulators being identified that are distinct from histone acetyltransferases and deacetylases .

What are the target synonyms and nomenclature variations for HIST1H2BC?

The HIST1H2BC protein has numerous synonyms and nomenclature variations in scientific literature, which researchers should be aware of when conducting literature searches or cross-referencing datasets. These variations include: H2BC4, H2BFL, HIST1H2BC (primary name), H2BC6, H2BFH, HIST1H2BE, H2BC7, H2BFG, HIST1H2BF, H2BC8, H2BFA, HIST1H2BG, H2BC10, H2BFK, and HIST1H2BI. Additionally, functional descriptive terms include: Histone H2B type 1-C/E/F/G/I, Histone H2B.1 A, Histone H2B.a, H2B/a, Histone H2B.g, H2B/g, Histone H2B.h, H2B/h, Histone H2B.k, H2B/k, Histone H2B.l, and H2B/l. The specific modification of crotonylation at lysine 12 is sometimes abbreviated as H2BK12cr in the literature. Understanding these naming conventions is essential for comprehensive literature searches and avoiding confusion when integrating data from different sources .

Which experimental applications are validated for Crotonyl-HIST1H2BC (K12) antibodies and what are the recommended dilutions?

Crotonyl-HIST1H2BC (K12) antibodies have been validated for multiple experimental applications, allowing researchers to investigate this histone modification across various research contexts. The specific applications and recommended dilution ranges are:

These recommended dilutions provide starting points for experimental optimization, but researchers should perform antibody titration experiments to determine optimal conditions for their specific cell lines, tissues, and experimental systems .

How should Western Blot experiments be designed and optimized for detecting Crotonyl-HIST1H2BC (K12)?

For optimal Western blot detection of Crotonyl-HIST1H2BC (K12), researchers should implement a specialized protocol that accounts for the unique properties of histones and their modifications. Begin with careful sample preparation: extract histones using acid extraction methods (typically with 0.2N HCl) to efficiently isolate histones from other cellular proteins. For best results, use fresh samples or properly preserved frozen samples with protease and histone deacetylase (HDAC) inhibitors to prevent modification loss during processing. Load 10-20μg of histone extract per lane on a high percentage (15-18%) SDS-PAGE gel to achieve proper separation of the relatively small histone proteins.

When transferring proteins, use PVDF membranes rather than nitrocellulose due to their better retention of small proteins. Block with 5% BSA in TBST rather than milk, as milk contains proteins that can cross-react with some histone antibodies. For primary antibody incubation, use dilutions between 1:100-1:1000 (optimized through titration experiments) and incubate overnight at 4°C. Include positive controls treated with sodium crotonylate (30mM for 4 hours has been validated) to confirm specificity, as demonstrated in multiple cell lines including HeLa, K562, and 293 cell lysates. For negative controls, consider using samples where the modification has been enzymatically removed or cells treated with inhibitors of crotonylation .

What considerations are important when designing ChIP experiments with Crotonyl-HIST1H2BC (K12) antibodies?

When designing Chromatin Immunoprecipitation (ChIP) experiments with Crotonyl-HIST1H2BC (K12) antibodies, several critical factors must be considered for successful outcomes. First, effective crosslinking is essential - use 1% formaldehyde for 10 minutes at room temperature for standard crosslinking, but optimize this time based on your cell type as excessive crosslinking can mask epitopes. For chromatin fragmentation, sonication parameters should be carefully optimized to achieve fragments between 200-500bp, which is ideal for histone modification ChIP.

Include appropriate controls in your experimental design: an input sample (pre-immunoprecipitation chromatin), an IgG control (non-specific antibody), and when possible, a positive control region known to be enriched for H2BK12cr and a negative control region lacking this modification. For immunoprecipitation, use 2-5μg of Crotonyl-HIST1H2BC (K12) antibody per ChIP reaction with chromatin from approximately 1-5×10^6 cells. Consider using magnetic beads rather than agarose for immunoprecipitation to reduce background and increase specificity.

After ChIP, analyze enrichment using qPCR, ChIP-seq, or other appropriate methods depending on your research question. For ChIP-qPCR, design primers for positive control regions (promoters of actively transcribed genes) and negative control regions (heterochromatin regions). For ChIP-seq, ensure sufficient sequencing depth (minimum 20 million reads) for proper coverage of histone modifications. Finally, consider the biological context of your experiment - crotonylation levels can be manipulated by treating cells with crotonylation inducers (like sodium crotonate) or by modulating the activity of enzymes that regulate crotonylation .

How do polyclonal and monoclonal Crotonyl-HIST1H2BC (K12) antibodies compare in research applications?

Polyclonal and monoclonal antibodies against Crotonyl-HIST1H2BC (K12) present distinct advantages and limitations that researchers should consider when selecting reagents for specific applications:

The search results indicate that both polyclonal (PACO59647) and recombinant monoclonal (EPR17593) antibodies are available for detecting Crotonyl-HIST1H2BC (K12). For applications requiring the highest specificity and reproducibility (such as genome-wide ChIP-seq studies), monoclonal antibodies are generally recommended. For applications where detection sensitivity is paramount, polyclonal antibodies may provide advantages. Each researcher should validate both types in their specific experimental system to determine which performs optimally for their particular application and cell/tissue type .

What is the relationship between crotonylation and other histone acylation modifications, and how can researchers study these comparatively?

Histone crotonylation belongs to a broader family of acylation modifications that includes acetylation, butyrylation, propionylation, and other more recently discovered acylations. These modifications share the common feature of neutralizing the positive charge on lysine residues, but differ in their hydrocarbon chain length, structure, and metabolism. Crotonylation is distinct due to its four-carbon chain with an unsaturated bond, creating a more rigid and bulkier modification compared to acetylation. This structural difference likely contributes to differential recognition by reader proteins and potentially distinct functional outcomes.

To study these modifications comparatively, researchers can implement several approaches. Mass spectrometry-based proteomics allows comprehensive profiling of different acylation types on the same histone residues. Sequential ChIP (re-ChIP) experiments can determine whether different acylation marks co-occur on the same histone molecules. Comparative ChIP-seq studies using antibodies against different acylation types (crotonylation, acetylation, etc.) at the same lysine residue (K12) can reveal genomic distribution patterns and potential functional differences. Researchers can also manipulate cellular metabolism to alter the availability of different acyl-CoA donors (acetyl-CoA, crotonyl-CoA) and observe effects on respective modifications.

Additionally, researchers can employ genetic or chemical approaches to modulate enzymes that preferentially regulate specific acylation types. When designing such comparative studies, it's crucial to use well-validated, modification-specific antibodies like the Crotonyl-HIST1H2BC (K12) and Acetyl-HIST1H2BC (K12) antibodies described in the search results, and to implement appropriate controls to confirm modification specificity .

What are common troubleshooting strategies for weak or non-specific signals when using Crotonyl-HIST1H2BC (K12) antibodies?

When encountering weak or non-specific signals with Crotonyl-HIST1H2BC (K12) antibodies, researchers should implement a systematic troubleshooting approach addressing several key aspects of their experimental protocol. For weak signals, consider the following strategies: increase antibody concentration by using a stronger dilution (e.g., move from 1:1000 to 1:500 for Western blots); extend primary antibody incubation time (overnight at 4°C instead of 1-2 hours); enhance signal detection using more sensitive substrates for Western blots or brighter fluorophores for immunofluorescence; and enrich for histone fractions using acid extraction to concentrate the target protein.

For non-specific signals, implement these strategies: increase blocking stringency by using 5% BSA instead of milk and extending blocking time to 2 hours; optimize washing steps by adding an extra wash and extending wash durations; reduce primary antibody concentration if background is high; include competitor peptides (unmodified H2BC K12 peptides) to confirm specificity; and validate with positive controls by treating cells with sodium crotonylate (30mM for 4 hours) to enhance the modification signal, as demonstrated in HeLa, K562, and 293 cell lines.

For both issues, consider sample preparation factors: use fresh samples when possible; include deacetylase/decrotonylase inhibitors (such as sodium butyrate or trichostatin A) during sample preparation to preserve modifications; and verify antibody storage conditions, as repeated freeze-thaw cycles may reduce antibody activity. Finally, compare results between different detection methods (e.g., if Western blot fails, try immunofluorescence) to determine if the issue is application-specific .

How should researchers properly store and handle Crotonyl-HIST1H2BC (K12) antibodies to maintain optimal activity?

Proper storage and handling of Crotonyl-HIST1H2BC (K12) antibodies is critical for maintaining their specificity and sensitivity over time. Based on manufacturer recommendations, these antibodies should be stored at -20°C for long-term storage in appropriately sized aliquots to minimize freeze-thaw cycles. The antibodies are typically supplied in a storage buffer containing 50% glycerol, 0.01M PBS at pH 7.4, and 0.03% Proclin 300 as a preservative. This formulation helps maintain antibody stability during freeze-thaw cycles, but repeated cycles should still be avoided.

When working with the antibody, thaw aliquots completely at room temperature or 4°C before use, and mix gently by inversion or light vortexing - avoid vigorous shaking which can cause protein denaturation. For daily use during experimental periods, small working aliquots can be stored at 4°C for up to one week, but should be returned to -20°C for longer periods. Always use clean, nuclease-free tubes and pipette tips when handling the antibody to prevent contamination.

Temperature fluctuations are particularly damaging to antibody quality, so avoid leaving the antibody at room temperature for extended periods. When shipping or transporting the antibody between laboratories, maintain cold chain conditions using dry ice or sufficient ice packs. Finally, regularly validate antibody performance using positive controls, especially after extended storage periods or if the antibody has been subjected to suboptimal conditions, to ensure experimental results remain reliable and reproducible .

How can Crotonyl-HIST1H2BC (K12) antibodies be integrated into multi-omics approaches to study epigenetic regulation?

Integrating Crotonyl-HIST1H2BC (K12) antibodies into multi-omics research strategies enables comprehensive analysis of histone crotonylation's role in chromatin regulation and gene expression networks. These antibodies serve as critical tools in several interconnected approaches: In ChIP-sequencing workflows, the antibodies facilitate genome-wide mapping of K12 crotonylation sites, which can be integrated with transcriptomic data (RNA-seq) to correlate modification patterns with gene expression profiles. When combined with ATAC-seq or DNase-seq data, researchers can investigate relationships between crotonylation and chromatin accessibility to understand how this modification influences DNA packaging and regulatory element exposure.

More sophisticated applications include CUT&RUN or CUT&Tag methodologies, which provide higher resolution mapping of crotonylation sites with improved signal-to-noise ratios compared to traditional ChIP-seq. Sequential ChIP (re-ChIP) approaches using antibodies against different histone modifications (first Crotonyl-K12, then another modification) can reveal co-occurrence patterns and hierarchical relationships between crotonylation and other epigenetic marks. For protein interaction studies, researchers can employ techniques like rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME) using Crotonyl-HIST1H2BC (K12) antibodies to identify proteins that specifically recognize or regulate this modification.

To study modification dynamics, researchers can combine these antibodies with nascent RNA sequencing methods like NET-seq or TT-seq to correlate crotonylation changes with transcription kinetics. Proximity ligation assays can also be performed to investigate spatial relationships between crotonylation and other chromatin features or nuclear structures. When designing such multi-omics studies, researchers must ensure batch consistency across different experimental platforms and implement appropriate computational integration strategies to derive meaningful biological insights from these complex datasets .

What is the role of Crotonyl-HIST1H2BC (K12) in disease contexts and how can the antibodies be used in translational research?

Histone crotonylation at H2BC-K12 is emerging as an important epigenetic modification with potential implications in various disease contexts, including cancer, inflammatory disorders, and metabolic diseases. Research suggests that aberrant crotonylation patterns may contribute to dysregulated gene expression in pathological states. In cancer research, Crotonyl-HIST1H2BC (K12) antibodies are valuable for comparative studies between normal and malignant tissues to identify altered crotonylation profiles associated with oncogenic programs. These antibodies can be applied in immunohistochemistry of patient-derived samples to evaluate whether crotonylation at this specific residue correlates with disease progression, treatment response, or patient outcomes.

For mechanistic studies, researchers can use these antibodies in cellular models where disease-relevant signaling pathways are activated or inhibited to determine how pathological processes affect crotonylation dynamics. ChIP-seq analyses in disease models can reveal genome-wide redistribution of H2BC-K12 crotonylation that may contribute to aberrant gene expression programs. The antibodies are also valuable for validating potential therapeutic approaches targeting enzymes that regulate crotonylation, such as histone crotonyltransferases or decrotonylases, which represent emerging targets for epigenetic therapies.

In inflammatory and metabolic disorders, these antibodies can help investigate how metabolic fluctuations (which affect crotonyl-CoA availability) impact histone crotonylation and subsequent gene regulation. There's particular relevance in studying conditions where short-chain fatty acid metabolism is altered, as these metabolites influence the cellular crotonylation landscape. Researchers should note that when using these antibodies in translational contexts, validation across multiple patient samples is essential to establish consistent patterns, and correlation with functional studies is needed to move beyond descriptive observations toward causal mechanisms .

How does cellular metabolism influence histone crotonylation patterns and what experimental approaches can measure these relationships?

Cellular metabolism and histone crotonylation are intricately connected through the availability of crotonyl-CoA, which serves as the substrate donor for histone crotonylation reactions. Several metabolic pathways influence crotonyl-CoA levels, including fatty acid β-oxidation, certain amino acid catabolism pathways (particularly lysine), and gut microbiome-derived short-chain fatty acids. To investigate these relationships experimentally, researchers can employ multiple complementary approaches using Crotonyl-HIST1H2BC (K12) antibodies as key detection tools.

Metabolic manipulation experiments provide direct insights: researchers can supplement cell culture media with crotonate (typically 2-30mM) or other short-chain fatty acids to artificially increase intracellular crotonyl-CoA pools and measure resulting changes in histone crotonylation using Western blotting or immunofluorescence with the Crotonyl-HIST1H2BC (K12) antibody. As demonstrated in validation studies, treatment with 30mM sodium crotonylate for 4 hours significantly increases detectable crotonylation in multiple cell lines including HeLa, K562, and 293 cells. Conversely, inhibiting fatty acid oxidation with compounds like etomoxir can reduce crotonyl-CoA availability and researchers can measure the impact on histone crotonylation levels.

Flux analysis using isotope-labeled metabolic precursors (13C-labeled crotonate or fatty acids) combined with mass spectrometry can track the incorporation of labeled carbons into histone crotonylation marks. Genetic manipulation of enzymes involved in crotonyl-CoA production or consumption pathways allows researchers to determine which metabolic routes most significantly impact histone crotonylation. ChIP-seq using Crotonyl-HIST1H2BC (K12) antibodies under different metabolic conditions (nutrient restriction, hypoxia, etc.) can reveal how metabolic states influence the genomic distribution of this modification.

For more comprehensive analysis, researchers can integrate metabolomics data (measuring crotonyl-CoA and related metabolites) with ChIP-seq or Western blot data using these antibodies to establish quantitative relationships between metabolite levels and histone modification patterns. When designing such studies, researchers should carefully control experimental conditions that might indirectly affect crotonylation, including cell density, serum composition, and the activity of deacylation enzymes like HDACs .