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) .
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 .
Cancer Progression:
Note: The FITC-conjugated variant offers cost-effective fluorescence detection compared to Alexa Fluor-linked antibodies.
Storage: Aliquot and store at -20°C; avoid freeze-thaw cycles .
Controls: Use knockout hepatocytes (KMT5C-deficient) to validate specificity .
Limitations: Not recommended for western blotting due to epitope accessibility .
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.
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:
Validation requires a three-step approach:
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.
Cross-reactivity testing: Use siRNA knockdown of homologous methyltransferases (e.g., KMT5A/B) to confirm absence of off-target binding .
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 |
The sequential staining method minimizes fluorophore crossover:
Primary staining: Apply FITC-KMT5C (1:500 in PBS/10% FBS) for 1 hr at RT
Secondary detection: Use Alexa Fluor 647-conjugated secondary antibodies
Critical wash step: 3× PBS + 0.1% Tween-20 between incubations
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% |
This common discrepancy (reported in 23% of studies ) arises from:
Technical factors:
Epitope accessibility differences in fixed (IF) vs denatured (WB) samples
Temporal expression variations during cell cycle progression
Biological factors:
Post-translational modifications affecting antibody affinity
Subcellular localization changes (nuclear vs cytoplasmic pools)
Troubleshooting protocol:
Synchronize cell cycles using double thymidine block
Compare multiple fixation methods (methanol vs paraformaldehyde )
Perform time-course analyses at 0, 6, 12, 24 hr post-treatment
The synergistic protocol involves:
Pharmacological inhibition: A196 (10 μM) pretreatment for 24 hr
Immune activation: Anti-PD-1 (10 mg/kg) administered q3d
Response monitoring:
Weekly PET-CT for metabolic tumor volume
Flow cytometry for CD8+ PD-1+ T cell frequencies
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% |
Implement a five-control system:
Isotype control: FITC-conjugated IgG at matched concentration
Input DNA: 2% of sonicated chromatin pre-immunoprecipitation
Competition control: 10× molar excess of non-conjugated KMT5C antibody
Epitope spike-in: Add 5% Drosophila S2 cell chromatin with exogenous KMT5C
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 |
The FITC conjugation enables real-time tracking with these modifications:
Quenching solution: 0.1 M glycine (pH 2.5) to neutralize unbound antibodies
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 |
A 2025-developed method enables:
FACS-based enrichment: Sort cells into KMT5C-high/-low populations via FITC intensity
gRNA sequencing: Amplify integrated sgRNAs from sorted populations
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 |
Mechanistic studies reveal:
A196 pretreatment ↑ olaparib sensitivity by 12-fold (IC50 shift from 1.2 μM → 0.1 μM)
Synergy arises from:
Impaired H4K20me2/3-dependent DNA repair
Persistent γH2AX foci (89% cells vs 22% control)
Mitotic catastrophe in 63% of treated cells
Experimental protocol for combination studies:
Pre-treat with A196 (5 μM, 24 hr)
Add PARPi (olaparib, 100 nM) for 48 hr
Assess viability via IncuCyte caspase-3/7 activation