2-hydroxyisobutyryl-HIST1H1C (K135) Antibody
Description
Introduction
The 2-hydroxyisobutyryl-HIST1H1C (K135) Antibody is a polyclonal rabbit antibody designed to detect the 2-hydroxyisobutyrylation modification at lysine 135 (K135) of histone H1.2 (encoded by HIST1H1C). This post-translational modification (PTM) is part of a broader class of lysine acylations that influence chromatin structure and gene regulation. The antibody is critical for studying epigenetic mechanisms, particularly in contexts where histone H1.2 plays a regulatory role in chromatin compaction, DNA methylation, and transcriptional silencing .
Immunogen and Target Details
The antibody targets HIST1H1C, a histone H1 variant critical for chromatin fiber assembly and gene silencing. Key properties include:
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Uniprot ID: P16403
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Synonyms: Histone H1.2, H1F2, H1c
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Modification: 2-hydroxyisobutyrylation at K135, a site linked to chromatin remodeling and transcriptional regulation .
ELISA
Used to quantify 2-hydroxyisobutyryl-HIST1H1C (K135) levels in lysates or purified histones. Dilution ranges typically span 1:2000–1:10,000 .
Immunofluorescence (IF)
Applied to detect subcellular localization of 2-hydroxyisobutyrylated HIST1H1C. For example:
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Protocol: Fixation with 4% formaldehyde, permeabilization with 0.2% Triton X-100, and blocking with 10% normal serum .
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Dilution: 1:10–1:100 for primary antibody, followed by Alexa Fluor 488-conjugated secondary antibody .
Cross-Reactivity and Specificity
No cross-reactivity data is explicitly reported, but the antibody is validated for human HIST1H1C. Its specificity for K135 2-hydroxyisobutyrylation is inferred from immunogen design .
Comparison with Other Antibodies
The K135 antibody differs from related variants (e.g., K158, K109) in applications and target specificity:
| Antibody | Target Site | Applications | Host | Reactivity |
|---|---|---|---|---|
| 2-hydroxyisobutyryl-HIST1H1C (K135) | K135 | ELISA, IF | Rabbit | Human |
| 2-hydroxyisobutyryl-HIST1H1C (K158) | K158 | ELISA, WB, ICC, ChIP | Rabbit | Human |
| 2-hydroxyisobutyryl-HIST1H1C (K109) | K109 | WB, ChIP | Rabbit | Human |
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- Research indicates that H1.2 influences the expression of a network of E2F target genes. It enhances the association of pRb with chromatin, strengthens transcriptional repression by pRb, and facilitates cell-cycle arrest in a pRb-dependent manner. (PMID: 28614707)
- BRG1, a chromatin remodeling protein, collaborates with H1.2 to repress gene expression. It facilitates H1.2 deposition and stabilizes nucleosome positioning near the transcription start site. (PMID: 27390128)
- Studies have revealed the presence of histones H1.2 and H1.4 in MDA-MB-231 metastatic breast cancer cells. Phosphorylation at specific residues within these histone variants (S173 of H1.2 and S172, S187, T18, T146, and T154 of H1.4) increases significantly during the M phase of the cell cycle, indicating a cell cycle-dependent regulation. Additionally, a SNP variant of H1.2 (A18V) has been observed in MCF-10A cells. (PMID: 26209608)
- Interactions between the C-terminal tail of linker histone isoforms and apoptotic intermediates suggest a broader function of these isoforms in apoptotic cascades. (PMID: 24525734)
- Post-translational modifications at T165 of H1.2 are not essential for chromatin binding or cell proliferation. However, modifications at K26 of H1.4 are crucial for proper cell cycle progression. (PMID: 24873882)
- H1.2 interacts with Cul4A and PAF1, promoting the activation of developmental regulatory genes. (PMID: 24360965)
- H1.2 is less abundant compared to other histone H1 variants at the transcription start sites of inactive genes. Promoters enriched in H1.2 are distinct from those enriched in other histone H1 variants and tend to be repressed. (PMID: 24476918)
- Mutations in linker histone genes HIST1H1 B, C, D, and E, as well as other genes like OCT2 (POU2F2), IRF8, and ARID1A, are implicated in the pathogenesis of follicular lymphoma. (PMID: 24435047)
- The p53 acetylation-H1.2 phosphorylation cascade acts as a unique mechanism for triggering p53-dependent DNA damage response pathways. (PMID: 22249259)
- Research has confirmed N-terminal acetylation on all histone H1 isoforms, along with a single internal acetylation site. Phosphorylation sites were identified within peptides containing the cyclin dependent kinase (CDK) consensus motif. (PMID: 15595731)
- The binding of histone H1 to a general amyloid-like motif suggests a common role for histone H1 in diseases associated with amyloid-like fibrils. (PMID: 16854430)
- Following treatment with bleomycin, histone H1.2 translocates from the nucleus to the mitochondria and colocalizes with Bak, a pro-apoptotic protein, within mitochondria. (PMID: 17879944)
- The recruitment of YB1, PURalpha, and H1.2 to the p53 target gene Bax is necessary for the repression of p53-induced transcription. (PMID: 18258596)
Q&A
What is 2-hydroxyisobutyryl-HIST1H1C (K135) Antibody?
2-hydroxyisobutyryl-HIST1H1C (K135) Antibody is a rabbit polyclonal antibody that specifically recognizes the 2-hydroxyisobutyryl modification at lysine 135 of the human Histone H1.2 protein (HIST1H1C). This antibody is generated using a peptide sequence surrounding the 2-hydroxyisobutyryl-Lys (K135) site derived from Human Histone H1.2. The immunogen consists of a synthetic peptide designed to elicit immune responses targeting this specific post-translational modification on histone H1.2. The antibody is unconjugated and belongs to the IgG isotype, making it suitable for various immunological detection methods .
What is the function of Histone H1.2 in chromatin structure?
Histone H1.2 (HIST1H1C) serves critical structural and regulatory functions in chromatin architecture. This linker histone binds to DNA between nucleosomes, facilitating the formation of the higher-order chromatin fiber structure. This interaction is essential for the condensation of nucleosome chains into more compact chromatin fibers, which influences genome packaging and accessibility. Beyond its structural role, Histone H1.2 acts as a regulator of individual gene transcription through multiple mechanisms including chromatin remodeling, nucleosome spacing, and DNA methylation processes .
What applications are recommended for 2-hydroxyisobutyryl-HIST1H1C (K135) Antibody?
The 2-hydroxyisobutyryl-HIST1H1C (K135) Antibody has been validated for several experimental applications:
| Application | Recommended Dilution | Notes |
|---|---|---|
| ELISA | 1:2000-1:10000 | For quantitative detection of the modified histone |
| Immunofluorescence (IF) | 1:20-1:200 | For cellular localization studies |
For immunofluorescence applications, the antibody has been successfully tested on HeLa cells treated with sodium butyrate, with cells fixed in 4% formaldehyde and permeabilized using 0.2% Triton X-100. This approach allows visualization of the specific histone modification within nuclear regions .
How do storage conditions affect 2-hydroxyisobutyryl-HIST1H1C (K135) Antibody performance?
Proper storage is critical for maintaining optimal antibody performance. The 2-hydroxyisobutyryl-HIST1H1C (K135) Antibody should be stored at -20°C or -80°C for long-term preservation. The antibody is typically supplied in a buffer containing 50% glycerol and 0.01M PBS at pH 7.4, with 0.03% Proclin 300 as a preservative. This formulation helps maintain antibody stability and activity. For -20°C storage, aliquoting is generally unnecessary due to the glycerol content, but for frequently accessed antibody stocks, creating small aliquots is advisable to avoid repeated freeze-thaw cycles that can compromise antibody quality and specificity .
What controls should be included when using 2-hydroxyisobutyryl-HIST1H1C (K135) Antibody?
When designing experiments with 2-hydroxyisobutyryl-HIST1H1C (K135) Antibody, several controls should be implemented to ensure valid and interpretable results:
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Blocking peptide control: Using the immunizing peptide to compete with the antibody binding can confirm specificity, as demonstrated in western blot analyses of histone antibodies where signal is eliminated in the presence of blocking peptide .
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Treatment controls: Cells treated with histone deacetylase inhibitors like sodium butyrate (as used in validation experiments) can serve as positive controls, enhancing the presence of histone modifications .
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Genetic knockdown/knockout controls: Samples with reduced expression of HIST1H1C can help validate antibody specificity for the target protein.
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Cross-reactivity assessment: Testing against related histone variants can confirm the antibody's specificity for HIST1H1C over other H1 subtypes .
How does Histone H1.2 interact with other chromatin components?
Histone H1.2 participates in complex interactions with various chromatin components to regulate genome organization and function. Research has revealed several important interaction patterns:
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Nucleosome binding: H1.2 binds to linker DNA regions between nucleosomes, with its globular domain contacting the DNA entry/exit points on the nucleosome.
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Polycomb complex interactions: Histone H1 has been linked with Polycomb complexes, suggesting a role in repressive chromatin formation. This connection was identified in chromatin-associated RNA samples but was not detected in mRNA preparations, highlighting the importance of experimental approach when studying these interactions .
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Transcriptional machinery interaction: H1.2 influences RNA polymerase II activity and positioning, affecting both coding and non-coding RNA production. Depletion of H1 can lead to altered RNA polymerase II distribution and activity patterns .
How does Histone H1 depletion affect genome stability and replication?
Research has uncovered critical connections between histone H1 levels and genome stability through replication processes. Depletion of histone H1 content results in widespread alterations to DNA replication mechanisms:
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Replication initiation disruption: Reducing histone H1 levels causes genome-wide alterations in replication initiation patterns, disrupting the normal temporal and spatial organization of origin firing .
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Replication fork stability: H1 depletion leads to significant fork stalling and decreased fork progression rates. DNA fiber analysis of human cells with knocked-down H1.2 and H1.4 showed marked decreases in fork rate and increases in fork asymmetry, indicating replication stress .
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DNA damage accumulation: The replication stress induced by H1 depletion results in increased DNA damage signaling, likely due to replication-transcription conflicts .
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Transcription-dependent effects: The replication defects observed in H1-depleted cells are transcription-dependent, as treatment with transcription inhibitors like DRB (5,6-dichlorobenzimidazole1-β-D-ribofuranoside) reverses both the decreased fork rate and increased fork asymmetry phenotypes .
What is the role of 2-hydroxyisobutyryl modification on K135 of HIST1H1C in transcriptional regulation?
The 2-hydroxyisobutyryl modification on lysine 135 of HIST1H1C represents an important post-translational modification that likely influences chromatin structure and gene expression. While the precise functional consequences of this specific modification at K135 are still being elucidated, several aspects have been established:
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Chromatin accessibility: Post-translational modifications of histone H1 generally alter its binding affinity to DNA, with modifications typically reducing H1-DNA interaction strength. The 2-hydroxyisobutyryl modification likely decreases the positive charge of the lysine residue, potentially reducing DNA binding affinity and promoting a more open chromatin state.
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Non-coding RNA regulation: Research has shown that histone H1 plays a role in regulating non-coding RNA turnover on chromatin. The triple knockout of H1 subtypes (including H1c, the mouse homolog of human HIST1H1C) resulted in the accumulation of thousands of chromatin-associated RNAs, suggesting H1's involvement in RNA processing and stability .
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Developmental regulation: In differentiated cells, knockdown of H1 variants leads to increased non-coding transcript association with chromatin, suggesting a conserved role for H1 in transcript regulation that may be influenced by specific modifications like 2-hydroxyisobutyrylation .
What methodological approaches can optimize immunofluorescence using 2-hydroxyisobutyryl-HIST1H1C (K135) Antibody?
For researchers conducting immunofluorescence studies with 2-hydroxyisobutyryl-HIST1H1C (K135) Antibody, several methodological considerations can enhance detection sensitivity and specificity:
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Cell treatment optimization: Pre-treating cells with 30mM sodium butyrate for 4 hours can enhance the detection of 2-hydroxyisobutyryl modifications by increasing their abundance, as demonstrated in the validation studies with HeLa cells .
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Fixation protocol: The recommended fixation protocol involves 4% formaldehyde followed by permeabilization with 0.2% Triton X-100. This approach preserves nuclear architecture while allowing antibody access to nuclear antigens .
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Blocking conditions: Implementing 10% normal goat serum in the blocking step helps reduce non-specific binding, particularly important when using secondary antibodies derived from goat .
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Antibody incubation: Overnight incubation at 4°C with the primary antibody (at 1:20-1:200 dilution) followed by appropriate fluorophore-conjugated secondary antibody (such as Alexa Fluor 488-conjugated AffiniPure Goat Anti-Rabbit IgG) provides optimal signal detection .
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Co-staining strategy: Combining 2-hydroxyisobutyryl-HIST1H1C (K135) Antibody with antibodies against other histone marks or nuclear proteins can provide valuable insights into the spatial relationship between different chromatin regulatory features.
