uev-3 Antibody
Description
Introduction to UEV-3 and Its Antibody
UEV-3 is a ubiquitin-conjugating enzyme variant that lacks the catalytic cysteine residue required for ubiquitin transfer but retains structural motifs critical for interactions within the ubiquitination machinery. In C. elegans, UEV-3 functions in the DLK-1 MAPK pathway, regulating synaptic architecture and axon termination by interacting with kinases like PMK-3 and MAK-2 . The UEV-3 antibody enables researchers to investigate its expression, localization, and mechanistic roles in neuronal development and disease models.
Development and Validation of UEV-3 Antibody
The commercially available anti-UEVLD antibody (Sigma-Aldrich, SAB4502309) is a rabbit-derived polyclonal antibody raised against a peptide corresponding to residues 141–190 of human UEVLD (UniProt accession: Q8IX28). Key validation data includes:
| Parameter | Specification |
|---|---|
| Host Species | Rabbit |
| Reactivity | Human |
| Applications | WB (1:500–1:1000), IHC (1:50–1:100), ELISA (1:40000) |
| Molecular Weight | ~52 kDa (antigen) |
| Immunogen Range | Amino acids 141–190 |
| Storage Conditions | -20°C in aqueous buffered solution |
This antibody has been validated for specificity in Western blotting and immunohistochemistry, with no cross-reactivity reported against related UEV proteins like UEV-1 or UEV-2 .
Role in Neuronal Development
UEV-3 operates cell-autonomously in neurons, as demonstrated in C. elegans studies where uev-3 loss-of-function suppressed synaptic defects in rpm-1 mutants. The antibody could localize UEV-3 to cytosolic and nuclear compartments in transgenic models, supporting its role in DLK-1 pathway regulation .
Interaction with MAP Kinases
UEV-3 binds the p38 MAPK PMK-3, potentially modulating substrate specificity or kinase activation. Co-immunoprecipitation assays using UEV-3 antibodies confirmed this interaction, particularly with catalytically inactive PMK-3 mutants .
Therapeutic Potential
UEV-3’s homologs in humans (UEVLD) are explored for roles in viral budding (e.g., Ebola VP40 protein) and cancer. Inhibitors targeting UEV domains, such as UEV-10 and UEV-11, reduce viral replication by disrupting ESCRT complex interactions .
Technical Considerations
-
Experimental Optimization:
-
Limitations:
Future Directions
UEV-3 antibodies could advance studies on:
Product Specs
**Constituents:** 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- UEV-3 may enhance the specificity of the DLK-1 pathway by contributing to the activation of PMK-3 or limiting the substrates accessible to PMK-3. PMID: 20592265
Q&A
Here’s a structured FAQ collection for researchers working with UEV-3 Antibody, incorporating experimental design considerations, data analysis challenges, and methodological guidance based on current literature:
Advanced Research Questions
-
How to resolve discrepancies in UEV-3 functional data across studies?
-
Case Analysis: In antiviral assays, UEV-3 showed no activity against EBOV/HIV-1 (IC50 >100 μM, Table 3) , despite its role in ubiquitination pathways. This suggests:
-
Strategy: Pair antibody-based UEVLD knockdown with rescue experiments using wild-type vs. mutant UEVLD constructs to isolate phenotype contributions.
-
-
Can UEV-3 antibody differentiate between UEVLD isoforms in cancer models?
-
Design: Combine IHC with isoform-specific qPCR (e.g., primers targeting exon 4–5 junction). Use siRNA targeting conserved regions to assess cross-isoform effects.
-
Data Interpretation: Prioritize cell lines with endogenous UEVLD overexpression (e.g., SW480 colon carcinoma) to minimize background noise.
-
-
How to investigate UEVLD’s role in polyubiquitination regulation using this antibody?
Mechanistic & Translational Questions
-
What explains UEV-3’s lack of antiviral activity despite targeting TSG101-UEV pathways?
-
Hypothesis: UEV-3 may stabilize TSG101-UEV conformations incompatible with viral budding (contrasting with destabilizing inhibitors like UEV-11) .
-
Testing: Perform DSF (differential scanning fluorimetry) with recombinant TSG101-UEV ± UEV-3 antibody to measure Tm shifts indicative of binding-induced stabilization/destabilization .
-
-
Can UEV-3 antibody be used to study ESCRT-independent roles of UEVLD?
Data Table: Key Functional Comparisons
| Parameter | UEV-3 Antibody | Small-Molecule UEV-3 |
|---|---|---|
| Effective Conc. | 1:500–1:2000 (WB) | >100 μM (Ineffective) |
| Toxicity (IC50) | N/A | >100 μM (A3R5,7 cells) |
| Pathway Specificity | UEVLD isoforms | TSG101-UEV destabilizer |
Methodological Notes
-
Multiplex Applications: Combine IF (1:50–1:200 dilution) with proximity ligation assays (PLA) to visualize UEVLD-protein interactions spatially.
-
CRITICAL: For functional studies, verify antibody lot-to-lot consistency using the same peptide immunogen (AA 141-190) .
-
Data Conflict Resolution: Cross-validate findings with orthogonal tools (e.g., de novo designed antibodies from JAM for epitope-specific targeting) .
