UBC16 Antibody
Description
Research Applications and Findings
UBE2W antibodies are instrumental in studying ubiquitination dynamics and their biological implications.
Role in DNA Damage Repair
UBE2W facilitates monoubiquitination of FANCD2 in the Fanconi anemia (FA) pathway, a critical step for resolving interstrand DNA crosslinks . This interaction is E3-dependent and specific to UV-induced damage rather than mitomycin C (MMC)-induced damage .
Protein Quality Control
In fission yeast (Schizosaccharomyces pombe), UBE2W (ortholog UBC16) regulates nuclear protein degradation via the ubiquitin-proteasome system (UPS). Deletion of ubc16 disrupts nuclear PQC, leading to accumulation of misfolded proteins .
Plant Stress Responses
In tomato, UBC16 homologs (e.g., SlUBC16) are implicated in stress responses, including pathogen defense. UBC16-mediated ubiquitination may modulate protein stability during stress adaptation .
Experimental Validation and Challenges
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Western Blot (WB): Optimized dilutions (1:500–1:1000) detect UBE2W in human and mouse lysates .
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ELISA: Used for quantitative analysis but requires experimental optimization .
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Immunofluorescence (IF): Confirms nuclear localization of UBE2W .
Challenges:
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Specificity: Polyclonal antibodies may exhibit off-target binding; monoclonal alternatives (e.g., H00055284-M01) address this .
Key Research Insights
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DNA Damage Signaling: UBE2W’s interaction with FANCL and FANCD2 highlights its role in FA pathway regulation .
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Protein Quality Control: UBE2W/UBC16 is required for nuclear UPS-mediated degradation, exemplified in yeast models .
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Evolutionary Conservation: Tomato UBC16 homologs suggest conserved roles in eukaryotic stress responses .
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- Human ubiquitin-conjugating enzyme (E2) shares homology with the Arabidopsis thaliana UBC-16 gene product. This homology is supported by the research article published in PubMed with the identifier PMID: 16368532.
Q&A
Here’s a structured FAQ collection for researchers working with UBC16 antibody, based on academic literature and experimental methodologies:
How to validate UBC16 antibody specificity in plant and mammalian systems?
Antibody validation requires multi-platform testing:
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Western Blot: Test against lysates from UBC16-knockout models (e.g., Arabidopsis mutants) .
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Immunofluorescence: Localize UBC16 to nuclei in mouse/human cells, consistent with its nuclear localization signals .
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Cross-reactivity: Confirm reactivity with human (UniProt: Q96B02), mouse, and Arabidopsis (UniProt: Q9SB51) homologs .
Key Data:
| Application | Species Reactivity | Supported Evidence | Source |
|---|---|---|---|
| Western Blot | Human, Mouse | Clear 18 kDa band in HEK293T | |
| Immunofluorescence | Arabidopsis | Nuclear signal in root cells |
How to resolve discrepancies in UBC16’s E3 ligase-independent activity reports?
Contradictions arise from:
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Assay conditions: Glycerol concentrations >5% artificially enhance activity .
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E3 co-factors: Bacterial effectors (e.g., AvrPtoB) nonspecifically boost activity .
Experimental Design Tips:
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Include negative controls without E1/E3 enzymes.
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Use catalytically inactive UBC16 (C87A mutation) to confirm activity is enzyme-dependent .
What are advanced applications of UBC16 antibodies in studying immune evasion?
UBC16 regulates immune signaling through:
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Proteasomal degradation: Targets immune receptors like FLS2 in plants .
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Pathogen interactions: Salmonella effector proteins exploit UBC16 to degrade host defense proteins .
Workflow:
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Co-immunoprecipitate UBC16 with pathogen effectors.
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Monitor substrate ubiquitination via tandem ubiquitin-binding entities (TUBEs) .
How to detect post-translational modifications of UBC16?
Use Phos-tag gels or mass spectrometry to identify phosphorylation sites. For ubiquitination:
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Express HA-tagged ubiquitin with UBC16 in Nicotiana benthamiana.
Why does UBC16 exhibit dual localization in cytoplasm and nucleus?
Its nuclear localization signals (NLS) at the C-terminal domain drive partial nuclear import. Validate via:
