PSMC5 Antibody, HRP conjugated
Description
Mechanistic Studies
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Proteasome Function: PSMC5 HRP-conjugated antibodies have been used to study proteasome dynamics, including protein homeostasis, cell cycle regulation, and DNA repair .
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Cancer Research: Elevated PSMC5 expression correlates with poor prognosis in colorectal cancer (CRC). Silencing PSMC5 suppresses CRC proliferation and metastasis by modulating immune infiltrates and EMT pathways .
Neurodevelopmental Disorders
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Pathogenic Variants: PSMC5 mutations (e.g., P320R) impair proteasome activity, leading to abnormal protein aggregation and synaptic defects. These alterations are linked to neurodevelopmental delays and intellectual disabilities .
Western Blot Performance
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Observed MW: 45–49 kDa (consistent with PSMC5’s predicted 46 kDa) .
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Sample Types: Validated in human cell lines (HEK-293, HeLa), mouse tissues, and rat brain lysates .
Immunohistochemistry
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Optimal Dilutions: 1:200–1:5000 for WB, 1:100–1:400 for IHC .
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Antigen Retrieval: Recommended with TE buffer (pH 9.0) or citrate buffer (pH 6.0) .
Oncology
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Biomarker Potential: High PSMC5 expression in CRC is associated with immune evasion (reduced CD8+ T cells) and increased macrophage/neutrophil infiltration .
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Therapeutic Target: PSMC5 inhibition enhances sensitivity to immune checkpoint blockade (e.g., anti-PD-L1) .
Neurology
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
PSMC5 is a component of the 26S proteasome, a multiprotein complex responsible for the ATP-dependent degradation of ubiquitinated proteins. This complex plays a crucial role in maintaining protein homeostasis by removing misfolded or damaged proteins that could compromise cellular function, and by eliminating proteins that are no longer needed. Consequently, the proteasome is involved in numerous cellular processes, including cell cycle progression, apoptosis, and DNA damage repair. PSMC5 is part of the heterohexameric ring of AAA (ATPases Associated with diverse cellular Activities) proteins. This ring unfolds ubiquitinated target proteins, simultaneously translocating them into a proteolytic chamber for degradation into peptides.
- The autoinflammation-associated H443P NLRC4 mutant exhibits altered interaction with SUG1 and ubiquitinated proteins, triggering constitutive caspase-8-mediated cell death dependent on FADD, but independent of Ser(533) phosphorylation. PMID: 27974463
- PSMC5 facilitates the damaging effects of radiation in radiation-responsive H460 cancer cells, suggesting its potential as a prognostic indicator for radiotherapy and molecular targeted therapy in lung cancer. PMID: 26592665
- XopJ exhibits protease activity, specifically targeting RPT6 for degradation. PMID: 25739698
- TRIP-1 regulates fibroblast acquisition of myofibroblast phenotype and function. PMID: 24528651
- γ-aminobutyric acid B receptor proteasomal degradation is mediated by the interaction of the GABAB2 C terminus with the proteasomal ATPase RPT6 and is regulated by neuronal activity. PMID: 24482233
- RPT6 directly interacts with CKIP-1 and promotes Smurf1 turnover. PMID: 23032291
- SUG1 plays a critical role in the transcription of MHC class I, and the MHC class II-like molecules, HLA-DM and HLA-DO. PMID: 22771340
- SUG1 is crucial for regulating histone H3K4me3 and H3R17me2 at the cytokine-inducible MHC-II and CIITA promoters. PMID: 19660582
- SUG1 participates in the ubiquitin/proteasome-mediated degradation of estrogen receptors. PMID: 14702340
- Proteasome dysfunction, induced by a proteasome inhibitor or siRNA-mediated Sug1 knockdown, upregulates MYO18B protein; MYO18B is polyubiquitinated in vivo. PMID: 16499872
- p45 plays a significant role in regulating ataxin-3 degradation by the proteasome. PMID: 17302910
- Both 19S and 20S subunits of the 26S proteasome play specific and critical roles in regulating CIITA activity and MHC class II transcription. PMID: 18215421
- The 19S ATPase SUG1 modifies histones to initiate major histocompatibility complex class II transcription, highlighting the proteasome's role in regulating mammalian transcription. PMID: 18662994
- SUG-1 uniquely links transcription and degradation processes through its interaction with SRC-3. PMID: 19144644
Q&A
How to Validate Specificity of HRP-Conjugated PSMC5 Antibodies in Western Blotting?
Methodological Approach:
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Positive/Negative Controls: Include lysates from tissues with high proteasome activity (e.g., human tonsil, mouse kidney) and PSMC5-knockdown cell lines .
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Blocking Peptide Assays: Pre-incubate the antibody with immunogen peptides (e.g., residues 127–406 of human PSMC5) to confirm signal loss .
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Cross-Species Validation: Test reactivity against human, mouse, and rat samples; PSMC5 antibodies often show cross-reactivity due to high sequence homology .
Data Interpretation:
| Sample Type | Predicted Band (kDa) | Observed Band (kDa) | Validation Outcome |
|---|---|---|---|
| Jurkat cells | 45 | 46 | Specific |
| Mouse kidney | 45 | 46 | Cross-reactive |
| PSMC5-KO cells | 45 | No band | Specificity confirmed |
What Experimental Designs Address PSMC5’s Role in Neurodevelopmental Proteasomopathies?
Advanced Workflow:
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Model Systems: Use Drosophila Rpt6 knockdown (ortholog of PSMC5) to study synaptic plasticity deficits . In mammalian systems, employ rat hippocampal neurons to quantify excitatory synapse loss via immunohistochemistry .
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Functional Assays: Measure proteasome activity using fluorogenic substrates (e.g., Suc-LLVY-AMC) in patient-derived cells with PSMC5 variants .
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Integrated Stress Response (ISR) Modulation: Treat cells with PKR/GCN2 kinase inhibitors to rescue immune and metabolic phenotypes .
Key Findings:
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PSMC5 loss-of-function increases protein aggregates by 3.2-fold in neurons .
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ISR inhibition restores mitophagy rates by 67% in patient cells .
How to Troubleshoot Non-Specific Staining in Immunohistochemistry?
Optimization Strategies:
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Antigen Retrieval: Boil formalin-fixed paraffin-embedded sections in sodium citrate buffer (pH 6.0) for 20 minutes .
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Blocking: Use 5% BSA with 0.1% Triton X-100 to reduce background .
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Titration: Test dilutions between 1:50 and 1:200; higher concentrations increase non-specific binding .
Case Example:
In human tonsil sections, optimal PSMC5 signal was achieved at 1:100 dilution with hematoxylin counterstaining .
How to Resolve Discrepancies in Observed vs. Predicted Molecular Weights?
Root-Cause Analysis:
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Post-Translational Modifications: PSMC5 undergoes phosphorylation (e.g., at Ser residues), increasing apparent molecular weight .
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Proteoform Detection: Use 2D gel electrophoresis to separate isoforms; HRP-conjugated antibodies may detect multiple bands .
Supporting Data:
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46 kDa band observed in human/mouse tissues aligns with phosphorylation status .
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Truncating variants (e.g., p.Asp394Glufs*2) produce lower-weight bands (~42 kDa) .
What Controls Are Essential for Knockdown/Overexpression Studies?
Required Controls:
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Rescue Experiments: Co-express wild-type PSMC5 in knockdown models to confirm phenotype reversibility .
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Proteasome Activity Assays: Compare chymotrypsin-like activity between PSMC5-deficient and control cells .
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qPCR Validation: Verify >70% mRNA reduction using primers spanning exon junctions .
Example Workflow:
| Step | Control Type | Outcome Metric |
|---|---|---|
| PSMC5 siRNA transfection | Scrambled siRNA | Proteasome activity (ΔRFU/min) |
| PSMC5 overexpression | Empty vector | Ubiquitinated protein levels (Western blot) |
How Does PSMC5 Antibody Cross-Reactivity Impact Multi-Species Studies?
Strategic Recommendations:
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Phylogenetic Alignment: Verify antibody epitope conservation (e.g., residues 253–324 are 89% conserved in human/mouse) .
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Species-Specific Lysates: Test antibody against rat muscle (46 kDa band expected) and zebrafish (non-reactive) .
Cross-Reactivity Table:
What Methodologies Quantify PSMC5’s Role in Protein Aggregation Diseases?
Advanced Techniques:
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Proximity Ligation Assay (PLA): Visualize PSMC5-ubiquitin colocalization in aggregates using Duolink® reagents .
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Cryo-EM: Resolve 26S proteasome structures in PSMC5 mutant cells to identify conformational changes .
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Lipidomics: Perform LC-MS on patient fibroblasts to quantify dysregulated phospholipids (e.g., +44% phosphatidylinositol) .
Data Correlation:
