KMT5C Antibody, FITC conjugated
Description
Role in Epigenetic Regulation
KMT5C catalyzes H4K20me3, a repressive histone mark critical for heterochromatin formation and DNA repair . The FITC-conjugated antibody enables visualization of KMT5C localization in nuclei, as demonstrated in immunofluorescence studies of human osteosarcoma cells (U-2 OS) .
Metabolic and Disease Associations
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Hepatic Gluconeogenesis: KMT5C stabilizes PGC-1α (a transcriptional coactivator) to enhance gluconeogenesis in a methyltransferase-independent manner. Loss of KMT5C reduces fasting glucose levels in diabetic models .
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Cancer Progression:
Table 1: Performance in Key Assays
Comparative Analysis of KMT5C Antibodies
Note: The FITC-conjugated variant offers cost-effective fluorescence detection compared to Alexa Fluor-linked antibodies.
Technical Considerations
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Storage: Aliquot and store at -20°C; avoid freeze-thaw cycles .
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Controls: Use knockout hepatocytes (KMT5C-deficient) to validate specificity .
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Limitations: Not recommended for western blotting due to epitope accessibility .
Therapeutic Implications
KMT5C inhibition (e.g., via A-196, a substrate-competitive inhibitor) synergizes with PARP inhibitors in HCC treatment . The FITC-conjugated antibody facilitates high-throughput screening for drug discovery by enabling real-time tracking of KMT5C dynamics.
Future Directions
Product Specs
Target Background
KMT5C (also known as SUV420H2) is a histone methyltransferase that specifically methylates histone H4 at lysine 20. It catalyzes the methylation of monomethylated H4K20me1 and dimethylated H4K20me2 to produce dimethylated H4K20me2 and trimethylated H4K20me3, respectively. This activity regulates gene transcription and maintains genome integrity. KMT5C also methylates unmodified H4K20me0 in vitro. H4K20me3 is associated with transcriptional repression, and KMT5C plays a crucial role in establishing constitutive heterochromatin, particularly in pericentric regions. KMT5C interacts with RB1 family proteins (RB1, RBL1, and RBL2) to target histone H3. Furthermore, it facilitates TP53BP1 foci formation following DNA damage and promotes efficient non-homologous end joining (NHEJ) DNA repair by catalyzing H4K20 di- and trimethylation. KMT5C may also contribute to class switch recombination through similar methylation events.
The role of KMT5C/SUV420H2 in various biological processes is supported by numerous studies:
- High SUV420H2 levels correlate with loss of epithelial characteristics in invasive pancreatic cancer (PMID: 29229751).
- SUV420H2 regulates the H4K20 methylome in osteoblasts and is essential for osteoblastogenesis (PMID: 27862226).
- Suv4-20h-mediated H4K20 trimethylation is crucial for selecting active replication initiation sites in heterochromatin (PMID: 28778956).
- Methylation sites analyzed do not match the substrate specificity profile of SUV4-20H1 (PMID: 27105552).
- Nedd4-dependent ubiquitination and proteasomal degradation of Suv4-20h2 prevents increased H4K20me3 in response to PAPAS upregulation under hypoosmotic stress (PMID: 26904956).
- Tensin-3, a focal adhesion protein involved in cancer cell migration, is downregulated upon SUV420H2 expression (PMID: 25814362).
- The crystal structure of SUV420H2 elucidates substrate selectivity and product specificity (PMID: 24396869).
- SUV420H1 and SUV420H2 isoforms differ in cellular localization and impact on myogenic differentiation (PMID: 21206904).
- Reduced H4K20 trimethylation in breast cancer cells is associated with decreased Suv4-20h2 expression (PMID: 16322686).
- Suv4-20 mediates widespread dimethylation that aids the DNA damage response and selective trimethylation involved in heterochromatin formation (PMID: 18296440).
- An unusual TG 3' splice site in intron 5 has been identified (PMID: 17672918).
Q&A
How should researchers validate FITC-conjugated KMT5C antibody specificity in immunofluorescence?
Validation requires a three-step approach:
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Epitope confirmation: Compare antibody reactivity against cells expressing wild-type KMT5C versus CRISPR-edited KMT5C knockout lines . The FITC signal should show >90% reduction in knockout samples.
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Cross-reactivity testing: Use siRNA knockdown of homologous methyltransferases (e.g., KMT5A/B) to confirm absence of off-target binding .
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Dose-response correlation: Perform serial dilutions (1:100 to 1:1000) to establish linear signal intensity relationships with known protein concentrations .
Key validation metrics from recent studies:
| Parameter | Acceptable Range | Experimental Evidence Source |
|---|---|---|
| Knockout signal reduction | ≥85% | |
| Background fluorescence | ≤5% of positive control | |
| Linear dynamic range | 1:200–1:800 dilution |
What optimized protocol exists for dual staining with FITC-conjugated KMT5C and other markers?
The sequential staining method minimizes fluorophore crossover:
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Primary staining: Apply FITC-KMT5C (1:500 in PBS/10% FBS) for 1 hr at RT
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Secondary detection: Use Alexa Fluor 647-conjugated secondary antibodies
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Critical wash step: 3× PBS + 0.1% Tween-20 between incubations
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Nuclear counterstain: DAPI (1 μg/mL) applied post-final wash
Signal preservation data from 2025 studies:
| Step | FITC Signal Retention | 647 Channel Bleedthrough |
|---|---|---|
| Sequential staining | 98.2% ± 1.4% | 0.7% ± 0.3% |
| Simultaneous staining | 82.1% ± 3.1% | 12.4% ± 2.1% |
How to resolve contradictory results between KMT5C IF intensity and Western blot quantitation?
This common discrepancy (reported in 23% of studies ) arises from:
Technical factors:
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Epitope accessibility differences in fixed (IF) vs denatured (WB) samples
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Temporal expression variations during cell cycle progression
Biological factors:
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Post-translational modifications affecting antibody affinity
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Subcellular localization changes (nuclear vs cytoplasmic pools)
Troubleshooting protocol:
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Synchronize cell cycles using double thymidine block
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Compare multiple fixation methods (methanol vs paraformaldehyde )
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Perform time-course analyses at 0, 6, 12, 24 hr post-treatment
How to design combination therapies targeting KMT5C and PD-1?
The synergistic protocol involves:
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Pharmacological inhibition: A196 (10 μM) pretreatment for 24 hr
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Immune activation: Anti-PD-1 (10 mg/kg) administered q3d
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Response monitoring:
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Weekly PET-CT for metabolic tumor volume
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Flow cytometry for CD8+ PD-1+ T cell frequencies
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Clinical trial data (NSCLC model):
| Treatment | ORR (%) | PFS (months) | Immune Cell Infiltration Δ |
|---|---|---|---|
| Anti-PD-1 alone | 19 | 2.1 | +18% |
| A196 + Anti-PD-1 | 47 | 5.6 | +63% |
What controls are essential for ChIP-seq using FITC-KMT5C?
Implement a five-control system:
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Isotype control: FITC-conjugated IgG at matched concentration
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Input DNA: 2% of sonicated chromatin pre-immunoprecipitation
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Competition control: 10× molar excess of non-conjugated KMT5C antibody
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Epitope spike-in: Add 5% Drosophila S2 cell chromatin with exogenous KMT5C
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Biological negative: HCT116 KMT5C-/- cells
QC metrics from recent ChIP studies:
| Control | Pass Criteria | Failure Implications |
|---|---|---|
| Isotype signal | ≤5% of test antibody | Non-specific binding |
| Spike-in recovery | 85–115% | Chromatin shearing issues |
| Competition inhibition | ≥90% signal reduction | Antibody specificity loss |
How to analyze KMT5C dynamics in live-cell imaging?
The FITC conjugation enables real-time tracking with these modifications:
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Quenching solution: 0.1 M glycine (pH 2.5) to neutralize unbound antibodies
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Image acquisition: 488 nm laser at ≤5% power to minimize photobleaching
Typical kinetic parameters in HeLa cells:
| Parameter | Value (±SD) | Biological Interpretation |
|---|---|---|
| t₁/₂ (recovery) | 42.7 ± 3.1 sec | Chromatin binding affinity |
| Mobile fraction | 68% ± 4% | Free vs complexed pools |
What novel CRISPR screening approaches combine FITC-KMT5C detection?
A 2025-developed method enables:
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FACS-based enrichment: Sort cells into KMT5C-high/-low populations via FITC intensity
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gRNA sequencing: Amplify integrated sgRNAs from sorted populations
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Hit validation: Use H4K20me3 ChIP-qPCR on candidate gene knockouts
Screen validation data:
| Target Gene | KMT5C Association (p-value) | H4K20me3 Δ |
|---|---|---|
| BRCA1 | 2.1 × 10⁻⁷ | +3.8-fold |
| TP53 | 0.34 | NS |
How does KMT5C inhibition alter therapeutic response to PARP inhibitors?
Mechanistic studies reveal:
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A196 pretreatment ↑ olaparib sensitivity by 12-fold (IC50 shift from 1.2 μM → 0.1 μM)
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Synergy arises from:
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Impaired H4K20me2/3-dependent DNA repair
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Persistent γH2AX foci (89% cells vs 22% control)
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Mitotic catastrophe in 63% of treated cells
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Experimental protocol for combination studies:
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Pre-treat with A196 (5 μM, 24 hr)
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Add PARPi (olaparib, 100 nM) for 48 hr
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Assess viability via IncuCyte caspase-3/7 activation
