UBLCP1 Antibody
Description
UBLCP1 Antibody Characteristics
The UBLCP1 antibody is a polyclonal rabbit IgG antibody that recognizes human, mouse, and rat UBLCP1. Key properties include:
| Property | Details |
|---|---|
| Target | UBLCP1 (UniProt ID: Q8WVY7) |
| Host Species | Rabbit |
| Reactivities | Human, mouse, rat |
| Applications | Western blot (WB), immunohistochemistry (IHC), immunofluorescence (IF/ICC), immunoprecipitation (IP), ELISA |
| Molecular Weight | 37 kDa (observed) / 37 kDa (calculated) |
| Immunogen | Fusion protein Ag2743 |
| Storage | PBS with 0.02% sodium azide and 50% glycerol; store at -20°C |
| Commercial Sources | Proteintech (12099-1-AP), Sigma-Aldrich (HPA039615) |
This antibody is affinity-purified and validated for specificity across multiple platforms, including the Human Protein Atlas .
Research Applications
UBLCP1 antibodies are widely used to explore proteasome regulation and nuclear protein degradation. Key applications include:
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Coimmunoprecipitation (IP): Demonstrates UBLCP1’s interaction with the 26S proteasome via its UBL domain and Rpn1 subunit .
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Subcellular Localization: Confirms UBLCP1’s exclusive nuclear localization, dependent on proteasome binding (Fig. 2C) .
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Functional Studies: RNAi-mediated UBLCP1 knockdown increases nuclear proteasome activity by enhancing regulatory particle (RP)-core particle (CP) assembly (Fig. 4B, 5D) .
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Post-Translational Modification Analysis: Identifies UBLCP1’s phosphatase activity toward Rpt1, a proteasome ATPase subunit, disrupting 26S proteasome assembly .
UBLCP1 as a Proteasome Phosphatase
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Mechanism: UBLCP1 dephosphorylates the 19S regulatory particle (RP), specifically targeting Rpt1 to impair ATPase activity and destabilize RP-CP interaction .
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Nuclear Specificity: UBLCP1 knockdown enhances nuclear proteasome activity by 30–50% but does not affect cytoplasmic activity (Fig. 4B) .
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Structural Dependence: The UBL domain is necessary for nuclear localization and proteasome binding, while phosphatase activity (DXDXT motif) is required for functional regulation .
Therapeutic Implications
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UBLCP1-deficient cells exhibit accelerated degradation of nuclear proteins (e.g., NLS-GFPu reporters), suggesting its role in diseases linked to proteasome dysregulation, such as cancer and neurodegenerative disorders .
Technical Considerations
Product Specs
Target Background
- Research indicates that UBLCP1 selectively binds to the 26S proteasome non-ATPase regulatory subunit 13 (19S regulatory particle) in regulating proteasome assembly. PMID: 28539385
- The high-resolution solution structure of the UBL domain of human UBLCP1 and its interaction with Rpn1 has been elucidated. PMID: 23667555
- UBLCP1 is a 26S proteasome phosphatase that regulates nuclear proteasome activity. PMID: 21949367
Q&A
How should researchers validate UBLCP1 antibody specificity for different experimental applications?
Validation requires a multi-step approach:
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Western Blot (WB): Confirm detection of a single band at 37 kDa in lysates from human brain tissue or K-562 cells, as demonstrated by Proteintech (12099-1-AP) . Parallel experiments with knockout cell lines (e.g., CRISPR/Cas9-edited UBLCP1) are critical to rule off-target effects.
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Immunohistochemistry (IHC): Use antigen retrieval buffers (TE pH 9.0 or citrate pH 6.0) for formalin-fixed paraffin-embedded (FFPE) tissues like ovary cancer samples . Compare staining patterns with negative controls lacking primary antibody.
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Functional validation: Co-immunoprecipitation (Co-IP) assays with proteasome subunits (e.g., Rpn1 or Rpt1) verify antibody utility in interaction studies .
What factors influence antibody dilution optimization for UBLCP1 detection?
Dilution ranges depend on:
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Sample type: Nuclear extracts require higher concentrations (1:50 for IHC in FFPE tissues) due to epitope masking.
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Assay sensitivity: Immunofluorescence (IF) in HeLa cells works optimally at 1:20–1:200 , while WB may require 1:500–1:2000 .
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Antigen abundance: Lower dilutions (1:20) are recommended for low-expression systems (e.g., neuronal cultures) . Titration curves using serial dilutions across biological replicates are mandatory .
How can researchers resolve discrepancies in subcellular localization studies of UBLCP1?
Conflicting reports about nuclear vs. cytoplasmic localization arise from:
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Fixation artifacts: Methanol fixation may leak nuclear antigens; paraformaldehyde with Triton X-100 permeabilization preserves nuclear signals .
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Cell cycle dependency: UBLCP1 relocates to proteasome-rich nuclear foci during S-phase . Synchronize cell populations using double thymidine block.
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Antibody cross-reactivity: Validate with siRNA knockdown controls (≥90% reduction) . A study using FLAG-tagged UBLCP1 confirmed nuclear colocalization with proteasomes .
What mechanistic insights can UBLCP1 antibodies provide about proteasome regulation?
UBLCP1 antibodies enable:
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Phosphatase activity profiling: Immunoprecipitated UBLCP1 dephosphorylates Rpt1 subunit (19S regulatory particle), reducing ATPase activity by 60% . Use Phos-tag gels to monitor Rpt1 phosphorylation states .
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Proteasome assembly assays: Native PAGE with nuclear fractions shows UBLCP1 depletion increases 26S proteasome abundance (2.3-fold vs. controls) . Combine with proteasome activity reporters (e.g., NLS-GFPu) .
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Disease modeling: Fibroblasts from ASD patients with UBLCP1 truncation mutations exhibit 40% higher chymotrypsin-like proteasome activity, reversible by MG132 inhibition .
How do researchers analyze UBLCP1’s role in neurodegenerative or oncological contexts?
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ALS models: Monitor TDP-43 aggregation in UBLCP1-deficient neurons; increased nuclear proteasome activity correlates with 30% faster degradation of misfolded proteins .
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Cancer studies: IHC in ovarian tumors shows UBLCP1 overexpression (2.5-fold vs. adjacent tissue) , linked to chemoresistance via proteasome suppression. Validate with salicylic acid-based inhibitors (e.g., compound 13, IC50 = 1.0 μM) .
| Experimental Design | Outcome Metrics |
|---|---|
| shRNA-mediated knockdown | ↑ Nuclear proteasome activity (1.8-fold) |
| Pharmacological inhibition | Rescue of Ub-G76V-GFP degradation (t1/2 = 45 min vs. 70 min controls) |
What strategies address contradictory data on UBLCP1-proteasome binding dynamics?
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Proteasome subcomplex isolation: Sucrose gradient centrifugation separates 19S (UBLCP1-bound) and 20S particles . Rpn1 co-IP confirms UBLCP1 interacts only with free 19S regulatory particles .
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Phosphomimetic mutants: Express Rpt1-S114D to bypass UBLCP1-mediated dephosphorylation, restoring 26S assembly in UBLCP1−/− cells .
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Live-cell imaging: FRET-based reporters (e.g., Rpn1-mCherry/UBLCP1-GFP) quantify interaction kinetics (koff = 0.18 s⁻¹) .
Can UBLCP1 antibodies distinguish between functional phosphatase domains and truncation mutants?
Yes, via:
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Epitope mapping: Proteintech’s antibody targets residues 150–250 , which are absent in exon 10 truncation mutants . Western blot shows truncated UBLCP1 at 28 kDa vs. 37 kDa wild-type .
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Functional assays: Truncated mutants fail to rescue proteasome hyperactivity in knockdown models (p < 0.01) . Use gentamicin to assess read-through efficiency .
