ung-1 Antibody
Description
Antibody Specificity and Validation
UNG-1 Antibody (e.g., Cat. No. 253636 from Abbiotec) is designed to distinguish UNG1 from UNG2. Validation data includes:
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Western Blot: Detects ~36 kDa band (predicted molecular weight) in mitochondrial lysates .
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Immunohistochemistry (IHC): Localizes UNG1 to mitochondria in human tissues (e.g., testis) .
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Cross-Reactivity: Species-specific reactivity confirmed in human, mouse, and rat . Notably, UNG1-specific antibodies show no cross-reactivity with UNG2 .
Validation Table
Applications in Research
UNG-1 Antibody is pivotal for:
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DNA Repair Studies: Investigating BER efficiency in mitochondrial DNA under oxidative stress .
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Cancer Research: Correlating UNG1 expression with mtDNA mutagenesis in aging and neurodegeneration .
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Immunodeficiency Models: Studying hyper-IgM syndrome type 5 (HIGM5), linked to UNG mutations .
Key Findings
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Aging: Decreased UNG1 activity in older mice correlates with elevated mtDNA damage .
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Disease Models: UNG-deficient B cells show impaired class switch recombination (CSR) and elevated genomic uracil .
Clinical Relevance
UNG1 dysfunction is implicated in:
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- The UNG-1 enzyme plays a critical role in DNA repair, specifically targeting damage induced by ionizing radiation. PMID: 20493785
- A cDNA encoding a Caenorhabditis elegans homologue (CeUng-1) of Escherichia coli Ung has been cloned and characterized. PMID: 18524757
Q&A
Here’s a structured collection of FAQs tailored for researchers working with UNG-1 antibody in academic settings, incorporating methodological insights and data from peer-reviewed studies:
What controls are essential for validating UNG-1 antibody specificity in WB/IHC?
Methodological Answer:
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Knockdown validation: Use siRNA targeting UNG in cell lines (e.g., HeLa) to confirm loss of signal .
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Competition assays: Pre-incubate antibody with recombinant UNG1 protein (10x molar excess) to block binding .
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Isoform-specific lysates: Test antibody on lysates from cells overexpressing UNG1 (mitochondrial) vs. UNG2 (nuclear) .
Key Finding: In UNG-mutant B cells (e.g., hyper-IgM patients), UNG-1 antibody detects residual uracil accumulation, confirming specificity .
How do I resolve contradictions in UNG-1 expression levels across tissue types?
Methodological Answer:
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Context-dependent analysis: UNG1 is highly expressed in post-mitotic tissues (e.g., heart), while UNG2 dominates in proliferating cells (e.g., activated B cells) .
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Cell cycle synchronization: Compare UNG1/2 levels in G0 vs. S-phase cells using flow cytometry-sorted populations .
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Quantitative PCR: Correlate mRNA levels (exon 1A vs. 1B) with WB data to rule out post-transcriptional regulation .
Example: In resting B cells, UNG1 constitutes 70% of total uracil-DNA glycosylase activity, but UNG2 becomes dominant after activation .
What advanced techniques leverage UNG-1 antibody to study somatic hypermutation (SHM) defects?
Methodological Answer:
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SHM sequencing: Combine UNG-1 IHC with IgH locus sequencing in UNG−/− B cells. Expect a 50–70% reduction in transversion mutations (C→G/A/T) due to repair defects .
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Error-prone repair assays: Use UNG-1 antibody to track uracil accumulation in MARs Eµ-deficient mice, which show aberrant SHM patterns .
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CRISPR interference: Knock out SMUG1 (backup glycosylase) in UNG-deficient models to assess functional redundancy .
Critical Data: In UNG-inhibited B cells, transitions (C→T) dominate (95% vs. 52% in controls), confirming UNG’s role in transversion generation .
How do I optimize UNG-1 antibody protocols for co-localization studies?
Methodological Answer:
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Fixation: Use 4% PFA (not methanol) to preserve mitochondrial/nuclear structures .
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Antibody titration: Test 2–20 µg/mL for IHC-P; higher concentrations (20 µg/mL) improve IF signal-to-noise .
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Cross-reactivity checks: Validate in UNG−/− mouse tissues (Thermo Fisher PA1-41024 cross-reacts with mouse/rat) .
Note: Murine UNG1 partially localizes to nuclei due to weaker mitochondrial targeting signals compared to human UNG1 .
How should I interpret conflicting data on UNG-1’s role in class switch recombination (CSR)?
Methodological Answer:
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Strain-specific analysis: Compare CSR efficiency in UNG−/− vs. AID−/− mice. UNG deficiency reduces CSR by ~60%, while AID ablation eliminates it .
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Uracil quantification: Use LC-MS/MS to measure genomic uracil levels. UNG-deficient cells show 3–5× higher uracil, impairing CSR .
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Pathway inhibition: Treat cells with Ugi (uracil glycosylase inhibitor) to mimic UNG loss and assess compensatory mechanisms .
Contradiction Resolution: SMUG1 cannot compensate for UNG2 in human B cells due to nuclear exclusion, explaining CSR defects in hyper-IgM patients .
