POLL Antibody
Description
Target Protein Overview
DNA polymerase lambda (POLL) is a 63.5 kDa protein encoded by the POLL gene in humans, consisting of 575 amino acids . Key characteristics include:
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Function: Participates in non-homologous end joining (NHEJ) and base excision repair (BER) pathways .
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Orthologs: Conserved in mice, rats, bovines, and zebrafish .
POLL Antibody Applications
POLL antibodies are validated for multiple experimental techniques:
Validation and Specificity Controls
To ensure antibody reliability, rigorous validation is required :
Recommended Controls
| Control Type | Purpose | Priority |
|---|---|---|
| Knockout (KO) tissue/cells | Confirm absence of nonspecific binding | High |
| Antigen pre-absorption | Block specific antibody binding | Medium |
| Positive tissue controls | Verify antibody-antigen recognition | High |
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Study Insights: A 2023 YCharOS study found that 50–75% of commercial POLL antibodies performed well in WB and IF, but ~12 publications per target used failing antibodies . Recombinant antibodies outperformed polyclonal/monoclonal variants in specificity .
Research and Disease Associations
POLL is implicated in multiple pathological and physiological contexts:
Disease Associations
| Disease/Condition | Publication Count | Key Role of POLL |
|---|---|---|
| Neoplasms | >180 | DNA repair dysfunction in cancers |
| Inflammation | >341 | Genomic stress response |
| Nervous System Disorders | >200 | Neuronal DNA damage repair |
Tissue Expression
| Tissue | Expression Level | Significance |
|---|---|---|
| Blood | High | Leukocyte DNA repair |
| Liver | Moderate | Metabolic stress response |
| Brain | Moderate | Neurodegeneration pathways |
Technical Considerations
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Epitope Retrieval: Citrate buffer (pH 6.0) recommended for FFPE tissues .
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Limitations: Commercial antibodies may exhibit batch variability; independent validation using KO controls is critical .
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Alternative Clones: Compare performance across vendors (e.g., Biocompare lists 2+ suppliers) .
Emerging Insights
Product Specs
Target Background
PMID: 21947619, Highlights the positive regulation of its expression by white light., .
PMID: 21325140, Shows that it recognizes 8-Oxo-G on a template as normal guanine and preferentially incorporates dCTP over dATP opposite this lesion., .
Q&A
What molecular functions of POLL necessitate specific antibody validation strategies?
POLL (DNA polymerase lambda) participates in base excision repair (BER) through its 5′-deoxyribose-5-phosphate lyase activity and strand-displacement synthesis . Its nuclear/chromosomal localization and involvement in short-/long-patch BER require antibodies validated for:
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Specific isoform detection: Human POLL has two isoforms (63–70 kDa) , necessitating antibodies distinguishing between them.
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Subcellular localization: Nuclear enrichment protocols should accompany antibody validation to confirm localization .
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Functional assays: Co-staining with repair markers (e.g., XRCC1) improves specificity .
How do polyclonal and monoclonal POLL antibodies differ in resolving technical vs. biological variability?
Methodological Insight: For BER pathway studies, combine polyclonal antibodies (broad epitope coverage) with siRNA knockdown controls to distinguish technical artifacts from biological variability .
What orthogonal validation methods are critical for POLL antibody specificity in DNA repair studies?
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Genetic knockout: Compare signal intensity in POLL-knockout vs. wild-type cell lines (e.g., HEK-293) .
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Recombinant protein controls: Use full-length POLL (63 kDa) and truncated variants to test cross-reactivity .
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IP-MS validation: Confirm co-precipitation with known interactors (e.g., PCNA) .
Data Contradiction Example: A 70 kDa band observed in WB may represent post-translationally modified POLL or cross-reactivity with POLB (DNA polymerase beta). Resolution requires:
How to optimize POLL antibody dilution for low-abundance targets in neuronal DNA damage models?
| Application | Recommended Dilution | Buffer Optimization |
|---|---|---|
| Western Blot | 1:1,000–1:5,000 | 5% BSA + 0.1% Tween-20 (reduce background) |
| Immunofluorescence | 1:100–1:500 | 0.3% Triton X-100 + 10% serum (permeabilization) |
Advanced Tip: For fixed brain tissue, antigen retrieval using citrate buffer (pH 6.0, 95°C, 20 min) improves epitope accessibility .
What computational tools aid in predicting POLL antibody cross-reactivity across species?
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Phylogenetic alignment: Compare immunogen sequence (e.g., human POLL residues 200–300) with orthologs (mouse: 85% identity; zebrafish: 64%) .
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Deep learning models: Train on antibody-antigen interaction datasets (e.g., SARS-CoV-2 spike protein studies ) to predict off-target binding.
Validation Workflow:
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In silico prediction using tools like BLAST and AlphaFold.
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Empirical testing in non-human models (e.g., mouse cortical neurons) .
How to resolve discrepancies in POLL expression data across cancer studies?
Reported POLL overexpression in breast vs. underexpression in lung cancers may stem from:
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Antibody clonality: Polyclonal antibodies may detect splice variants overexpressed in specific contexts .
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Repair pathway activation: Transient POLL upregulation during chemotherapy-induced DNA damage .
Methodological Solution:
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Use time-course experiments with genotoxic agents (e.g., H₂O₂).
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Normalize to housekeeping proteins with similar turnover rates (e.g., histone H3) .
What controls are essential for ChIP-seq using POLL antibodies?
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Input DNA normalization: Account for chromatin accessibility biases .
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Functional negative control: Cells treated with PARP inhibitor (suppresses BER recruitment) .
Data Interpretation: Peak enrichment at known BER loci (e.g., APEX1) confirms specificity .
