CCPG1 Antibody
Description
Antibody Characteristics
Key Observations:
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The rabbit polyclonal antibody (13861-1-AP) offers broad reactivity (human, mouse, rat) and compatibility with multiple assays, making it versatile for basic research .
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The mouse monoclonal antibody (60641-2-PBS) is optimized for conjugation-free applications like ELISA and multiplex assays .
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Abcam’s rabbit polyclonal (ab219854) is validated for ICC/IF, with a focus on human samples .
Western Blot (WB)
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Proteintech (13861-1-AP): Detects a band at ~110 kDa in HepG2, L02, and THP-1 cells. A verified customer noted background bands but confirmed specificity via siRNA knockdown .
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Cell Signaling (80158): Targets endogenous CCPG1 with predicted sensitivity for 105–120 kDa proteins .
Immunohistochemistry (IHC)
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Proteintech (13861-1-AP): Validated in human stomach cancer tissue with antigen retrieval using TE buffer (pH 9.0) or citrate buffer (pH 6.0) .
Immunofluorescence (IF/ICC)
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Abcam (ab219854): Effective for intracellular staining of human samples, with immunogen mapped to aa 250–400 .
ELISA and Multiplex Assays
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Proteintech (60641-2-PBS): Part of a matched antibody pair (MP50919-1/60641-2) for cytometric bead arrays .
Role in ER-Phagy
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CCPG1 acts as a bispecific ER-phagy receptor, recognizing ER luminal proteins (e.g., 6xIAPP, P3H4) and autophagosomal membranes .
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It contains cargo-interacting regions (CIRs) that bind distinct ER-resident cargos, enabling simultaneous recognition of multiple targets .
Autophagy Machinery Interactions
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Binds ATG8 family proteins (LC3/GABARAP) via a canonical LIR motif and FIP200 through a discrete motif .
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Knockdown studies reveal its essential role in clearing ER-luminal protein aggregates, preventing UPR hyperactivation .
Subcellular Localization
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Localizes to the perinuclear ER and peripheral ER foci, with autophagy flux increasing focus abundance under stress .
Antibody Applications in Functional Studies
Product Specs
Target Background
- CCPG1, an ER-resident protein, has been identified as a non-canonical cargo receptor that directly binds to core autophagy proteins. This binding occurs through an LIR motif to mammalian ATG8 proteins and, independently and via a discrete motif, to FIP200. PMID: 29290589
Q&A
How should researchers validate CCPG1 antibody specificity in different experimental systems?
Validation requires parallel approaches:
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Western Blot (WB): Use lysates from cell lines with confirmed CCPG1 expression (e.g., HepG2, L02, THP-1) and compare bands to the expected molecular weight (87 kDa calculated vs. 110 kDa observed) . Post-translational modifications or alternative splicing may explain discrepancies. Include knockout controls if available.
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Immunohistochemistry (IHC): Optimize antigen retrieval with TE buffer (pH 9.0) or citrate buffer (pH 6.0) . Validate using human stomach cancer tissues, where CCPG1 is enriched.
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Immunofluorescence (IF): Confirm perinuclear ER localization in HepG2 cells . Co-stain with ER markers (e.g., Calnexin) for spatial validation.
Table 1: Validation Data for CCPG1 Antibody (13861-1-AP)
| Application | Validated Cell Lines/Tissues | Recommended Dilution | Key Controls |
|---|---|---|---|
| WB | HepG2, L02, THP-1 | 1:500–1:1000 | Knockout lysates, peptide blocking |
| IHC | Human stomach cancer | 1:50–1:500 | Isotype controls, tissue-negative controls |
| IF/ICC | HepG2 | 1:200–1:800 | ER marker co-staining |
Why does CCPG1 exhibit a higher observed molecular weight (110 kDa) than calculated (87 kDa)?
The discrepancy arises from:
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Post-translational modifications: Phosphorylation or ubiquitination .
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Alternative splicing: Murine studies reveal isoforms affecting migration .
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Protein complexes: CCPG1 binds Dbs and Cdc42, potentially forming stable complexes detectable via WB .
Methodological Recommendation: Perform deglycosylation assays (e.g., PNGase F treatment) and compare migration patterns across species (human, mouse, rat) .
What factors influence antibody performance in detecting CCPG1 across applications?
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Dilution optimization: Titrate antibodies empirically (e.g., 1:50–1:500 for IHC vs. 1:200–1:800 for IF) .
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Fixation methods: Methanol fixation preserves epitopes better for IF than paraformaldehyde in some cases .
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Buffer compatibility: Use PBS-based buffers for storage to maintain IgG stability .
How do subcellular localization discrepancies of CCPG1 impact data interpretation?
CCPG1 localizes to perinuclear ER and peripheral foci under stress , but overexpression or fixation artifacts may alter observations. For example:
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Overexpression artifacts: Full-length CCPG1 may aggregate, while ΔTransm mutants show diffuse ER patterns .
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Stress conditions: ER-phagy induction (e.g., thapsigargin treatment) redistributes CCPG1 to autophagosomal membranes .
Methodological Recommendation: Use confocal microscopy with deconvolution (e.g., Zeiss Axiovert systems) and validate findings with siRNA knockdown .
How does CCPG1 regulate ER-phagy, and how can this be interrogated experimentally?
CCPG1’s LIR domain binds LC3/GABARAP, recruiting autophagosomes to stressed ER . Key approaches:
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Functional assays: Combine CCPG1 siRNA (65% knockdown efficacy ) with ER stress inducers (e.g., tunicamycin). Monitor ER-phagy via TEM or GFP-LC3 puncta quantification.
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Co-immunoprecipitation (Co-IP): Validate LC3 interactions using crosslinking agents (e.g., DSP) to stabilize transient complexes .
What experimental strategies resolve contradictions in CCPG1’s interaction with Rho GTPases?
CCPG1 scaffolds Dbs to modulate RhoA/Cdc42 activity, but cell-type-specific effects occur :
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In COS7 cells: Endogenous CCPG1 inhibits Dbs-mediated RhoA activation .
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In NIH 3T3 cells: Low CCPG1 levels permit RhoA activation.
Methodological Recommendation:
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Use cell lines with varying CCPG1 expression (e.g., COS7 vs. NIH 3T3) .
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Employ RhoGEF activity assays (e.g., G-LISA) alongside Co-IP to map context-dependent interactions.
Table 2: CCPG1-Dbs Interaction Mapping
| Ccpg1 Deletion Mutant | Dbs Binding | RhoA Activation |
|---|---|---|
| Full-length | Yes | Inhibited |
| Δ311 | No | Unaffected |
| ΔTransm | Yes | Partial inhibition |
