Crotonyl-HIST1H4A (K8) Antibody
Description
Definition and Biological Context
Crotonyl-HIST1H4A (K8) antibodies target histone H4 proteins modified by crotonylation at lysine 8. Histone crotonylation involves the addition of a crotonyl group (derived from crotonyl-CoA) to lysine residues, altering chromatin structure and gene expression . This PTM is implicated in transcriptional activation, DNA repair, and diseases like cancer .
Key Features of Histone H4 Crotonylation:
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Structural Role: Modifies nucleosome dynamics, influencing DNA accessibility .
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Regulatory Function: Part of the "histone code" that recruits chromatin-modifying enzymes .
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Disease Relevance: Dysregulation observed in acute kidney injury (AKI) and cancer .
Antibody Development and Validation
The Crotonyl-HIST1H4A (K8) antibody (e.g., ab201075, ab251336, CAC15418) is a rabbit-derived recombinant monoclonal antibody validated across multiple platforms:
Table 1: Validation Data
Specificity Controls:
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Negative Controls: No signal observed with secondary antibody alone .
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Cross-Reactivity: No binding to unmodified or unrelated histone peptides (e.g., acetylated H4K8) .
3.1. Mechanistic Studies
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Chromatin Remodeling: Used to study crotonylation's role in transcriptional regulation. For example, Gcn5 and Esa1 complexes were identified as crotonyltransferases using similar antibodies .
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Disease Models: Detects increased histone crotonylation in AKI, correlating with nephroprotective gene expression (e.g., PGC1α, Sirt-3) .
3.2. Technical Applications
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Western Blotting: Detects crotonylated H4 in 10 µg of whole-cell lysates .
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ChIP-Seq: Validated for chromatin immunoprecipitation to map crotonylation sites genome-wide .
Key Research Findings
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Crotonylation vs. Acetylation: Unlike acetylation, crotonylation is associated with distinct transcriptional outcomes, potentially due to its larger hydrophobic moiety .
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Therapeutic Insights: Crotonate supplementation in AKI models increases histone crotonylation, reducing inflammation and preserving renal function .
Limitations and Considerations
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Species Specificity: Limited to human, mouse, and rat samples .
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Antibody Competition: Cross-reactivity with other acylations (e.g., acetylation) has not been fully ruled out .
Future Directions
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- Studies indicate that PP32 and SET/TAF-Ibeta proteins inhibit HAT1-mediated H4 acetylation. PMID: 28977641
- Research suggests that post-translational modifications of histones, trimethylation of lysine 36 in H3 (H3K36me3) and acetylation of lysine 16 in H4 (H4K16ac), are involved in DNA damage repair. H3K36me3 stimulates H4K16ac upon DNA double-strand breaks. SETD2, LEDGF, and KAT5 are essential for these epigenetic changes. (SETD2 = SET domain containing 2; LEDGF = lens epithelium-derived growth factor; KAT5 = lysine acetyltransferase 5) PMID: 28546430
- Data show that Omomyc protein co-localizes with proto-oncogene protein c-myc (c-Myc), protein arginine methyltransferase 5 (PRMT5), and histone H4 H4R3me2s-enriched chromatin domains. PMID: 26563484
- H4K12ac is regulated by estrogen receptor-alpha and is associated with BRD4 function and inducible transcription PMID: 25788266
- Systemic lupus erythematosus seems to be linked to an imbalance in histone acetyltransferases and histone deacetylase enzymes, favoring pathological H4 acetylation. PMID: 25611806
- Sumoylated human histone H4 inhibits chromatin compaction by hindering long-range internucleosomal interactions. PMID: 25294883
- Acetylation at lysine 5 of histone H4 is associated with lytic gene promoters during reactivation of Kaposi's sarcoma-associated herpesvirus. PMID: 25283865
- An increase in histone H4 acetylation caused by hypoxia in human neuroblastoma cell lines corresponds to increased levels of N-myc transcription factor in these cells. PMID: 24481548
- Data suggest that G1-phase histone assembly is limited to CENP-A and H4. PMID: 23363600
- This study focused on the distribution of a specific histone modification, namely H4K12ac, in human sperm and characterized its specific enrichment sites in promoters throughout the entire human genome. PMID: 22894908
- SRP68/72 heterodimers function as major nuclear proteins whose binding of the histone H4 tail is inhibited by H4R3 methylation. PMID: 23048028
- TNF-alpha inhibition of AQP5 expression in human salivary gland acinar cells is attributed to an epigenetic mechanism involving suppression of acetylation of histone H4. PMID: 21973049
- Findings indicate that global histone H3 and H4 modification patterns are potential markers of tumor recurrence and disease-free survival in non-small cell lung cancer PMID: 22360506
- HAT1 differentially impacts nucleosome assembly of H3.1-H4 and H3.3-H4. PMID: 22228774
- Phosphorylation of histone H4 Ser 47, catalyzed by the PAK2 kinase, promotes nucleosome assembly of H3.3-H4 and inhibits nucleosome assembly of H3.1-H4 by enhancing the binding affinity of HIRA to H3.3-H4 and reducing the association of CAF-1 with H3.1-H4 PMID: 21724829
- The imatinib-induced hemoglobinization and erythroid differentiation in K562 cells are associated with global histone H4 PMID: 20949922
- Research reveals the molecular mechanisms by which the DNA sequences within specific gene bodies are sufficient to nucleate the monomethylation of histone H4 lysine 200, which, in turn, reduces gene expression by half. PMID: 20512922
- Downregulated by zinc and upregulated by docosahexaenoate in a neuroblastoma cell line. PMID: 19747413
- Low levels of histone acetylation are associated with the development and progression of gastric carcinomas, possibly through alterations in gene expression PMID: 12385581
- Overexpression of MTA1 protein and acetylation levels of histone H4 protein are closely correlated. PMID: 15095300
- Peptidylarginine deiminase 4 regulates histone Arg methylation by converting methyl-Arg to citrulline and releasing methylamine. Data suggest that PAD4 mediates gene expression by regulating Arg methylation and citrullination in histones PMID: 15345777
- The lack of biotinylation of K12 in histone H4 is an early signaling event in response to double-strand breaks PMID: 16177192
- Incorporation of acetylated histone H4-K16 into nucleosomal arrays inhibits the formation of compact 30-nanometer-like fibers and hinders the ability of chromatin to form cross-fiber interactions PMID: 16469925
- Apoptosis is associated with global DNA hypomethylation and histone deacetylation events in leukemia cells. PMID: 16531610
- BTG2 contributes to retinoic acid activity by promoting differentiation through a gene-specific modification of histone H4 arginine methylation and acetylation levels. PMID: 16782888
- A relationship exists between histone H4 modification, epigenetic regulation of BDNF gene expression, and long-term memory for extinction of conditioned fear. PMID: 17522015
- The H4 tail and its acetylation play novel roles in mediating the recruitment of multiple regulatory factors that can alter chromatin states for transcription regulation PMID: 17548343
- Brd2 bromodomain 2 is monomeric in solution and dynamically interacts with H4-AcK12; additional secondary elements in the long ZA loop may be a common characteristic of BET bromodomains. PMID: 17848202
- Spermatids Hypac-H4 impairment in mixed atrophy was not further deteriorated by AZFc region deletion. PMID: 18001726
- The SET8 and PCNA interaction couples H4-K20 methylation with DNA replication PMID: 18319261
- H4K20 monomethylation and PR-SET7 are critical for L3MBTL1 function PMID: 18408754
- High expression of acetylated H4 is more prevalent in aggressive than indolent cutaneous T-cell lymphoma. PMID: 18671804
- Findings suggest an essential role of histone H4 modifications in bronchial carcinogenesis PMID: 18974389
- Results indicate that by acetylation of histone H4 K16 during S-phase, early replicating chromatin domains acquire the H4K16ac-K20me2 epigenetic label that persists on the chromatin throughout mitosis and is deacetylated in early G1-phase of the next cell cycle PMID: 19348949
- Acetylated H4 is overexpressed in diffuse large B-cell lymphoma and peripheral T-cell lymphoma relative to normal lymphoid tissue. PMID: 19438744
- The release of histone H4 by holocrine secretion from the sebaceous gland might play a significant role in innate immunity. PMID: 19536143
- Histone modification, including PRC2-mediated repressive histone marker H3K27me3 and active histone marker acH4, might be involved in CD11b transcription during HL-60 leukemia cell reprogramming to terminal differentiation PMID: 19578722
- A role of Cdk7 in regulating elongation is further suggested by enhanced histone H4 acetylation and diminished histone H4 trimethylation on lysine 36 - two marks of elongation - within genes when the kinase was inhibited. PMID: 19667075
- Data demonstrated the dynamic fluctuation of histone H4 acetylation levels during mitosis, as well as acetylation changes in response to structurally distinct histone deacetylase inhibitors. PMID: 19805290
- Data directly implicate BBAP in the monoubiquitylation and additional posttranslational modification of histone H4 and an associated DNA damage response. PMID: 19818714
Q&A
What is the Crotonyl-HIST1H4A (K8) Antibody and what epitope does it recognize?
The Crotonyl-HIST1H4A (K8) Antibody is a polyclonal antibody raised in rabbits against a peptide sequence surrounding the crotonylated lysine 8 (K8) residue of human histone H4 (HIST1H4A). This antibody specifically recognizes the post-translational modification of crotonylation at the K8 position of histone H4, one of the core histone proteins that comprise the nucleosome. The immunogen used for antibody production is a peptide sequence around the site of Crotonyl-Lys (8) derived from Human Histone H4 . This specificity allows researchers to detect this particular modification in various experimental applications including ELISA, Western blotting (WB), immunocytochemistry (ICC), immunofluorescence (IF), and chromatin immunoprecipitation (ChIP) .
How does histone H4K8 crotonylation differ from other histone modifications?
Histone H4K8 crotonylation involves the addition of a crotonyl group to the lysine 8 residue of histone H4. Unlike more common modifications such as acetylation or methylation, crotonylation features a larger, more hydrophobic modification with a distinctive structure that includes a C=C double bond. This structural difference results in unique biological functions and protein interactions. While acetylation typically neutralizes the positive charge of lysine and promotes open chromatin states, crotonylation creates a more substantial modification that may recruit specific reader proteins distinct from those that recognize acetylation. H4K8 crotonylation is one of several crotonylation sites identified on different histones, including HIST1H3A (K18), HIST1H3A (K4), HIST1H3A (K9), and HIST1H4A (K5) .
Which applications has the Crotonyl-HIST1H4A (K8) Antibody been validated for?
The Crotonyl-HIST1H4A (K8) Antibody has been validated for multiple research applications that are essential for epigenetic studies. According to product specifications, this antibody has been verified for use in ELISA (Enzyme-Linked Immunosorbent Assay), which allows for quantitative detection of the modification in protein samples . It has also been validated for Western blotting (WB), enabling researchers to detect the presence and relative abundance of H4K8 crotonylation in cell and tissue lysates . For cellular localization studies, the antibody has been confirmed effective in immunocytochemistry (ICC) and immunofluorescence (IF), allowing visualization of the modification within cellular contexts . Importantly, it has been validated for chromatin immunoprecipitation (ChIP), a crucial technique for investigating the genomic distribution of histone modifications and their association with specific DNA sequences . This range of validated applications makes the antibody a versatile tool for comprehensive studies of H4K8 crotonylation in epigenetic research.
What are the optimal protocols for using Crotonyl-HIST1H4A (K8) Antibody in ChIP experiments?
For optimal chromatin immunoprecipitation with the Crotonyl-HIST1H4A (K8) Antibody, careful attention to experimental conditions is essential. The recommended protocol begins with proper chromatin preparation by crosslinking cells with formaldehyde, followed by cell lysis and sonication to generate chromatin fragments of appropriate size (200-500 bp). For the immunoprecipitation step, researchers should use approximately 10 μg of chromatin and an antibody dilution of 1:50 . This ratio has been validated to provide optimal enrichment while minimizing background. The antibody-chromatin mixture should be incubated overnight at 4°C with rotation, followed by addition of protein A/G magnetic beads. After thorough washing to remove non-specific binding, the crosslinks can be reversed and the DNA purified for subsequent analysis. Including appropriate controls, such as IgG or input samples, is critical for validating the specificity of the immunoprecipitation.
How should Crotonyl-HIST1H4A (K8) Antibody be used in Western blotting applications?
For effective Western blotting with the Crotonyl-HIST1H4A (K8) Antibody, histone extraction protocols are critical. Acid extraction methods are recommended to enrich for histones from cellular samples. After protein quantification, 5-10 μg of histone extract should be loaded per lane on a 15% SDS-PAGE gel to achieve optimal separation of the relatively small histone proteins. Following transfer to a PVDF membrane, blocking should be performed with 5% BSA in TBST. The Crotonyl-HIST1H4A (K8) Antibody should be used at a 1:1000 dilution and incubated overnight at 4°C . It is crucial to include deacetylase and decrotonylase inhibitors in all buffers to preserve the modification. Researchers should verify specificity using appropriate controls such as crotonylation-depleted samples or peptide competition assays, as cross-reactivity with acetylation marks can sometimes occur.
What are the considerations for using Crotonyl-HIST1H4A (K8) Antibody in immunofluorescence experiments?
For successful immunofluorescence with the Crotonyl-HIST1H4A (K8) Antibody, cell fixation and permeabilization protocols significantly impact epitope accessibility. The recommended procedure involves fixing cells with 4% paraformaldehyde followed by permeabilization with Triton X-100. For immunostaining, the antibody should be used at a dilution range of 1:200 to 1:500 and incubated overnight at 4°C . This dilution range provides optimal signal-to-noise ratio for detecting the H4K8 crotonylation mark within cellular contexts. When analyzing results, researchers should be aware that the distribution pattern of H4K8 crotonylation may vary significantly between cell types and in response to different cellular states or treatments affecting metabolic pathways. For dual staining with other histone mark antibodies, considerations regarding antibody compatibility based on host species and implementation of proper controls to ensure specificity are essential.
How can Crotonyl-HIST1H4A (K8) Antibody be integrated into genome-wide studies?
For genome-wide mapping of H4K8 crotonylation, ChIP-seq with the Crotonyl-HIST1H4A (K8) Antibody provides comprehensive insights into the distribution of this modification across the genome. The antibody has been specifically validated for ChIP applications, making it suitable for this advanced technique . When designing such experiments, sufficient sequence depth is crucial—researchers should aim for at least 20 million uniquely mapped reads per sample. For bioinformatic analysis, specialized peak-calling algorithms optimized for histone modifications should be used rather than those designed for transcription factor binding sites. To interpret the biological significance of identified enrichment patterns, integration with other genomic datasets including RNA-seq, accessibility assays, and other histone modifications is recommended. This approach can reveal patterns of co-occurrence and mutual exclusivity with other epigenetic marks, providing insights into the regulatory functions of H4K8 crotonylation.
What experimental approaches can distinguish the specific effects of H4K8 crotonylation from other histone modifications?
To isolate the specific effects of H4K8 crotonylation from other histone modifications, researchers should implement a multi-faceted approach. First, utilizing site-specific histone mutants where lysine 8 is replaced with residues that either mimic crotonylation or prevent it in appropriate model systems can provide direct functional evidence. Second, employing CRISPR-based epigenome editing by fusing catalytically inactive Cas9 with crotonyl writers or erasers directed to specific genomic loci can establish causality between the modification and observed phenotypes. Third, modulating cellular crotonyl-CoA levels through metabolic interventions while monitoring effects on H4K8cr levels and gene expression can reveal the interplay between metabolism and epigenetic regulation. Finally, conducting competition assays with recombinant nucleosomes containing specifically modified histones can identify proteins that preferentially bind to H4K8cr versus other modifications at the same residue, elucidating specific downstream effectors.
How does the specificity of Crotonyl-HIST1H4A (K8) Antibody compare to other crotonylation antibodies?
The specificity of the Crotonyl-HIST1H4A (K8) Antibody should be considered in relation to other available crotonylation antibodies. As shown in the comparative data below, this antibody specifically recognizes the K8 position of histone H4, while related antibodies target different lysine residues on histones H3 and H4.
| Antibody | Target Residue | Host | Clonality | Validated Applications | Species Reactivity |
|---|---|---|---|---|---|
| Crotonyl-HIST1H4A (K8) | Lysine 8 (H4) | Rabbit | Polyclonal | ELISA, WB, ICC, IF, ChIP | Human |
| Crotonyl-HIST1H4A (K5) | Lysine 5 (H4) | Rabbit | Polyclonal | ELISA, WB, IF, IP, ChIP | Human |
| Crotonyl-HIST1H3A (K18) | Lysine 18 (H3) | Rabbit | Polyclonal | ELISA, IF, ChIP | Human |
| Crotonyl-HIST1H3A (K9) | Lysine 9 (H3) | Rabbit | Polyclonal | ELISA, WB, ICC, IP, ChIP | Human |
This comparison highlights that while all these antibodies target crotonylation modifications, each recognizes a specific lysine residue in a distinct sequence context . When designing experiments to study crotonylation patterns, researchers should consider using multiple antibodies to gain a comprehensive understanding of the distribution and function of different crotonylation sites.
What are common issues with Crotonyl-HIST1H4A (K8) Antibody specificity and how can they be addressed?
Common specificity issues with the Crotonyl-HIST1H4A (K8) Antibody include potential cross-reactivity with other acyl modifications on the same residue (particularly acetylation) or crotonylation at similar sequence contexts on different histones. To address these concerns, researchers should implement multiple validation approaches. Peptide competition assays using crotonylated, acetylated, and unmodified H4K8 peptides can confirm specificity. Western blots with recombinant histones bearing defined modifications can directly assess cross-reactivity. Comparing samples from cells treated with HDAC inhibitors versus crotonate supplementation can determine if the antibody discriminates between acetylation and crotonylation. Additionally, validation with an alternative Crotonyl-HIST1H4A (K8) Antibody from a different manufacturer or raised against a different epitope region can provide further confidence in observed results. For definitive analysis in critical experiments, orthogonal approaches such as mass spectrometry should be considered to independently confirm the presence and abundance of H4K8 crotonylation.
How should researchers interpret contradictory results between different detection methods for H4K8 crotonylation?
When facing contradictory results between different methods detecting H4K8 crotonylation, researchers should implement a systematic analytical approach. First, the strengths and limitations of each methodology should be evaluated: ChIP detects genomic localization but may be affected by antibody specificity issues; Western blotting provides a global view but may miss site-specific dynamics; mass spectrometry offers absolute identification but may have sensitivity limitations for low-abundance modifications. Second, biological variables that could explain genuine differences should be considered, such as cell-type specificity, dynamic temporal changes, or response to unrecognized environmental factors. Third, technical variables should be examined, including extraction protocols, fixation methods, or buffer compositions that might affect the preservation of the modification. Implementing method-specific controls and combinatorial approaches where multiple methods target the same biological question from different angles can help resolve apparent contradictions and reveal the true biological complexity of H4K8 crotonylation dynamics.
What recommended experimental conditions optimize detection with Crotonyl-HIST1H4A (K8) Antibody?
Optimal detection with the Crotonyl-HIST1H4A (K8) Antibody requires careful attention to experimental conditions across different applications. The table below summarizes recommended parameters for various techniques:
| Application | Sample Preparation | Antibody Dilution | Incubation Conditions | Critical Controls |
|---|---|---|---|---|
| Western Blot | Acid extraction of histones | 1:1000 | Overnight at 4°C | Peptide competition, crotonylation-depleted samples |
| Immunofluorescence | 4% PFA fixation, 0.2% Triton X-100 permeabilization | 1:200-1:500 | Overnight at 4°C | Secondary-only control, pre-immune serum |
| ChIP | 1% formaldehyde crosslinking, sonication to 200-500bp | 1:50 | Overnight at 4°C | IgG control, input sample (10%) |
| ChIP-seq | As above, plus library preparation | As above | As above | Input sequencing, IgG ChIP-seq |
| ELISA | Histone coating at 1-10 μg/ml | 1:1000-1:5000 | 2 hours at RT | Unmodified peptide, competitive ELISA |
Across all applications, inclusion of deacetylase and decrotonylase inhibitors (such as sodium butyrate, trichostatin A, and nicotinamide) in buffers is essential to preserve the modification during sample processing . Additionally, researchers should be aware that storage conditions and freeze-thaw cycles can affect antibody performance, so aliquoting the antibody and following manufacturer-recommended storage protocols is advisable.
