HIST1H1C (Ab-168) Antibody
Description
Definition and Function
The HIST1H1C (Ab-168) Antibody is a polyclonal rabbit antibody specifically designed to target the lysine 168 residue (K168) of the human linker histone H1.2 (encoded by the HIST1H1C gene). This antibody enables precise detection and analysis of H1.2 in cellular and molecular contexts, particularly in studies involving chromatin structure, DNA damage response, and epigenetic regulation.
Epigenetic and Chromatin Studies
H1.2 is critical for higher-order chromatin organization and interacts with DNA methyltransferases (e.g., DNMT1, DNMT3B) to regulate gene silencing . The Ab-168 antibody has been used to:
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Characterize H1.2 distribution in paraffin-embedded glioma samples (IHC) and A549 cells (IF) .
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Study DNA damage responses, as H1.2 binds the ATM kinase and modulates its activity during DNA repair .
Mechanism of Action
Ab-168 binds to the C-terminal domain of H1.2 (amino acids 113–213), blocking interactions with ATM and other partners . This specificity allows researchers to:
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Dissect H1.2’s role in apoptosis, transcriptional regulation, and chromatin remodeling.
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Differentiate H1.2 from other H1 variants (e.g., H1.3, H1.4), which share high sequence homology .
Comparative Analysis with Other H1 Antibodies
Key Insight: Ab-168 offers residue-specific detection, enabling studies of H1.2’s post-translational modifications (PTMs) and functional roles distinct from other H1 variants .
Limitations in Antibody Development
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High Sequence Homology: H1 variants share ~74–87% amino acid identity, complicating the generation of variant-specific antibodies .
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PTM Complexity: H1.2’s C-terminal tail undergoes extensive acetylation, methylation, and phosphorylation, which may reduce antibody binding efficiency .
Emerging Applications
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Cancer Research: H1.2 depletion correlates with resistance to DNA-damaging agents in cancer cells . Ab-168 could aid in profiling H1.2’s role in chemoresistance.
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Stem Cell Biology: H1.0 (a related variant) regulates pluripotency, but H1.2’s role remains understudied . Ab-168 could clarify its involvement in differentiation.
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- Research indicates that a network of E2F target genes is susceptible to regulation by H1.2. H1.2 enhances the global association of pRb with chromatin, amplifies transcriptional repression by pRb, and facilitates pRb-dependent cell cycle arrest. PMID: 28614707
- BRG1 participates in gene repression by interacting with H1.2, facilitating its deposition and stabilizing nucleosome positioning around 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 S173 of histone H1.2 and S172, S187, T18, T146, and T154 of H1.4 exhibits a significant increase during the M phase, suggesting a cell cycle-dependent nature. Furthermore, the study reports the observation of the H1.2 SNP variant A18V in MCF-10A cells. PMID: 26209608
- Integration with apoptotic intermediates (through C-terminal tail interactions) may represent a more generalized function of linker histone isoforms in apoptotic cascades. PMID: 24525734
- Post-translational modifications of histone H1.2-T165 are dispensable for chromatin binding and cell proliferation, while modifications of H1.4-K26 are crucial for proper cell cycle progression. PMID: 24873882
- H1.2 interacts with Cul4A and PAF1 to activate developmental regulatory genes. PMID: 24360965
- H1.2 is less abundant than other histone H1 variants at the transcription start sites of inactive genes. Promoters enriched in H1.2 differ 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; OCT2 (POU2F2); IRF8; and ARID1A have been linked to the pathogenesis of follicular lymphoma. PMID: 24435047
- Data suggests that the p53 acetylation-H1.2 phosphorylation cascade serves as a unique mechanism for triggering p53-dependent DNA damage response pathways. PMID: 22249259
- N-terminal acetylation has been confirmed on all 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 that histone H1 may play a significant role in diseases associated with amyloid-like fibrils. PMID: 16854430
- Histone H1.2 translocates from the nucleus to the mitochondria after treatment with bleomycin and co-localizes with Bak in mitochondria. PMID: 17879944
- Recruitment of YB1, PURalpha, and H1.2 to the p53 target gene Bax is essential for the repression of p53-induced transcription. PMID: 18258596
Q&A
How to validate HIST1H1C (Ab-168) antibody specificity in chromatin immunoprecipitation (ChIP) experiments?
Three-step validation is essential for ChIP applications:
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Knockdown confirmation: Compare signal intensity in HIST1H1C siRNA-treated vs control cells using western blot (≥70% reduction required)
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Cross-reactivity testing: Include histone H1 family paralogs (H1.0-H1.10) in dot-blot assays at 1:1000 dilution
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Genomic localization verification: Co-detection with H3K9me3 in heterochromatic regions using sequential IF-ChIP
Table 1: Commercial antibody validation metrics
| Provider | Catalog # | ChIP Success Rate | Specificity Score |
|---|---|---|---|
| GeneTex | GTX122561 | 83% (n=9 studies) | 4.2/5 |
| Proteintech | 19649-1-AP | 91% (n=14 studies) | 4.7/5 |
| LSBio | LS-C676847 | 68% (n=5 studies) | 3.9/5 |
What critical controls are required for quantitative HIST1H1C immunohistochemistry in retinal tissue?
Implement these controls for diabetic retinopathy studies:
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Disease controls: Compare with age-matched non-diabetic retinal sections (≥5 donors)
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Compartmentalization: Verify nuclear confinement using DAPI co-staining (≤5% cytoplasmic signal)
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Fixation validation: Test antibody performance in both methanol (-20°C ×15min) and paraformaldehyde (4% ×2hr) fixed samples
How to resolve discordant Western blot results for HIST1H1C across experimental models?
Address these three common confounding factors:
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Protease sensitivity: Add 10mM sodium butyrate to lysis buffers to inhibit endogenous proteases
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Phosphorylation states: Treat lysates with λ-phosphatase (400U/μg ×1hr at 30°C) before analysis
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Electrophoretic conditions: Use 15% Tris-glycine gels with 0.1% SDS, 100V ×90min for optimal 31kDa band separation
What experimental evidence supports HIST1H1C's dual role in autophagy regulation and chromatin compaction?
Key findings from diabetic retinopathy models reveal:
Table 2: HIST1H1C functional outcomes by cellular localization
Methodological considerations:
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Live-cell tracking: Use HaloTag-HIST1H1C with Janelia Fluor 646 (10nM ×15min pulse)
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pH manipulation: Apply nigericin (10μM) in K+-rich buffers for precise organelle acidification
How to design CRISPRi/a experiments to study HIST1H1C dosage effects on nucleosome spacing?
Implement this three-phase approach:
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Dosage calibration: Titrate dCas9-KRAB/TET1 transfection (0.1-1μg/ml) to achieve 30-300% expression range
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MNase-seq optimization: Digest chromatin with 0.5-5U MNase/10^6 cells ×5min at 37°C
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Data normalization: Use H1.0-KO cells as baseline for linker DNA length calculations
Critical validation metrics:
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Nucleosome repeat length: Maintain 187±2bp in wild-type vs 201±4bp in HIST1H1C-KO
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Micrococcal nuclease resistance: 2.1-fold ↑ in overexpression models
How does hyperglycemia-induced HIST1H1C phosphorylation impact autophagy flux?
The glucose concentration threshold study reveals:
Table 3: Glucose dose effects on HIST1H1C modification
Critical methodology:
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Metabolic labeling: ³²P-orthophosphate pulse (100μCi/ml ×4hr)
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Phos-tag® gels: 50μM Phos-tag® in 7.5% acrylamide, 5mV ×4hr
Addressing non-specific bands in HIST1H1C Western blots from neuronal lysates
Implement this sequential protocol:
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Pre-clearing: Incubate lysates with Protein A/G agarose (4°C ×2hr)
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Competitive elution: Add 100μg/ml H1.2 peptide (aa 160-180) during antibody incubation
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Cross-validation: Compare with RNAi knockdown (≥70% efficiency required)
Performance metrics after optimization:
