Histone H4K20me3 Antibody
Description
Definition and Biological Context
Histone H4K20me3 antibodies are monoclonal or polyclonal immunoreagents designed to specifically recognize the trimethylated lysine 20 residue on histone H4. This modification is enriched in heterochromatin, telomeres, and repetitive DNA elements, where it facilitates chromatin compaction, suppresses transposable elements, and maintains genomic integrity . H4K20me3 is catalyzed by methyltransferases like KMT5C (Suv4-20h2) and interacts with DNA repair machinery and chromatin remodelers .
Table 1: Key Validation Metrics for H4K20me3 Antibodies
Chromatin Profiling
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CUT&RUN: High-efficiency recovery of H4K20me3-enriched regions, even with low cell inputs (e.g., 50k cells) .
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ChIP: Used to map H4K20me3 at heterochromatin and retrotransposons in embryonic stem cells .
Functional Studies
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Genomic Stability: Antibodies reveal H4K20me3’s role in repressing LINE/LTR elements during differentiation .
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Reprogramming: Loss of H4K20me3 correlates with chromatin relaxation in induced pluripotent stem cells, enhancing telomerase access .
Technical Considerations
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Cross-Reactivity: Some antibodies may detect H4K20me2, necessitating optimization of blocking buffers (e.g., 0.05% Tween 20) .
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Sample Preparation: High-salt/sonication protocols are recommended for Western blotting due to chromatin-bound protein insolubility .
Research Insights
Product Specs
Q&A
Validating Antibody Specificity for H4K20me3 in Chromatin Studies
Experimental Design:
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Peptide Competition Assays: Pre-incubate antibodies (1 µg) with 10x molar excess of methylated (H4K20me3) vs. unmethylated peptides for 1 hr at 4°C. Test residual immunoreactivity via dot blot .
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Cross-Reactivity Screening: Use recombinant histones with defined PTMs (e.g., H4K20me1/2) in Western blots. Active Motif’s validation data shows <5% cross-reactivity with H4K20me2 under optimized conditions .
Optimizing H4K20me3 Antibody Dilution for Diverse Applications
Methodological Framework:
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CUT&RUN: Start with 1:100 dilution (0.5 µg/µl stock) in digitonin buffer .
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Immunofluorescence: Titrate from 1:50 to 1:200 in PBS-Triton X-100. Note: Over-concentration causes pericentromeric smearing .
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Western Blot: Use 1:500–1:2000 dilutions with HDAC inhibitors (e.g., 5 mM sodium butyrate) to enhance signal .
Critical Parameter:
"Antibody performance inversely correlates with fixation time. Limit formaldehyde fixation to 10 min for ChIP assays to preserve H4K20me3 epitopes" .
Addressing Species Reactivity Limitations in Cross-Model Studies
Case Study:
While EpiCypher’s monoclonal antibody detects human and C. elegans H4K20me3 , polyclonal variants (Abbexa, Antibodies.com) show broader reactivity in murine systems . For non-model organisms:
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Perform epitope alignment using Clustal Omega to identify sequence conservation around K20.
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Validate via parallel IF staining with H4K20me3-deficient cell lines (e.g., Suv4-20h1/2 KO MEFs) .
Resolving Contradictory H4K20me3 Localization Data in Viral vs. Endogenous Chromatin
Context: HSV-1 latency studies show H4K20me3 absence at viral promoters despite strong centromeric association .
Resolution Strategy:
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Chromatin Accessibility Controls: Compare MNase digestion profiles between viral episomes and host chromatin.
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Spike-In Normalization: Use Drosophila S2 chromatin with known H4K20me3 levels as internal control .
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Multiplexed CUT&RUN: Co-target H3K27me3 to distinguish facultative heterochromatin domains .
Data Interpretation Table:
| Genomic Region | H4K20me3 Signal (Fold vs. IgG) | H3K27me3 Co-Occurrence |
|---|---|---|
| HSV-1 LAT Promoter | 1.2±0.3 | 4.8±0.6 |
| Human Centromere | 18.9±2.1 | 1.1±0.2 |
| C. elegans Germline | 9.4±1.7 | Not Detected |
Integrating H4K20me3 Data with DNA Methylation Profiling
Workflow:
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Sequential ChIP-MS: Perform H4K20me3 ChIP followed by MBD-Seq to map DNAme-H4K20me3 co-regulated regions.
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Computational Integration: Use Bismark + MACS3 pipelines to align 5mC and H4K20me3 peaks.
Key Finding:
"Co-occurrence at LINE-1 elements is 73% higher than random expectation (P < 0.001, Fisher’s exact test), suggesting synergistic silencing mechanisms" .
Time-Course Analysis of H4K20me3 Dynamics During Cell Cycle
Innovative Approach:
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AbFlex Recombinant Antibodies: Engineer clone 6F8-D9 with LPXTG motif for sortase-mediated Alexa Fluor 647 conjugation .
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Sequential Staining:
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Fix with 4% PFA (10 min) → H4K20me3 detection
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Methanol permeabilization (-20°C, 5 min) → γH2AX staining
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Validation: Correlation coefficient (r) = 0.89 between H4K20me3 and 53BP1 foci in irradiated cells .
Methodological Recommendations Table
| Challenge | Solution | Validation Metric |
|---|---|---|
| Low signal in IF | Antigen retrieval (10 mM citrate, 95°C) | Pericentromeric foci clarity |
| Cross-reactivity with H4K20me2 | Pre-adsorb with 50 µg/ml H4K20me2 peptide | Dot blot intensity ratio >25:1 |
| ChIP background noise | Dual-barcode spike-in controls | RPKM CV <15% across replicates |
