CPOX Antibody
Description
Definition and Overview of CPOX Antibody
CPOX antibody is a specific immunological reagent designed to detect coproporphyrinogen III oxidase (CPOX), a mitochondrial enzyme critical in heme biosynthesis. CPOX catalyzes the oxidative decarboxylation of coproporphyrinogen III to protoporphyrinogen IX, a key step in producing heme for hemoglobin and cytochrome proteins . Mutations in the CPOX gene are linked to hereditary coproporphyria, an autosomal dominant disorder characterized by acute neurological attacks and photosensitivity .
CPOX antibodies are categorized into monoclonal (e.g., B-9, PAT36B10AT) and polyclonal (e.g., rabbit-derived) variants, each optimized for specific applications such as Western blotting (WB), immunoprecipitation (IP), immunofluorescence (IF), and enzyme-linked immunosorbent assay (ELISA) . These antibodies are validated for human, mouse, and rat samples, with species-specific reactivity noted in product specifications .
Applications and Validation of CPOX Antibodies
Critical Research Findings:
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CPOX in Brain Tumors: Elevated CPOX mRNA and protein levels correlate with strong 5-ALA–induced fluorescence in malignant gliomas, aiding photodynamic diagnosis .
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Mouse Models: Cpox-deficient mice (e.g., BALB.NCT-Cpox) exhibit systemic coproporphyrin accumulation, mimicking human hereditary coproporphyria .
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Species-Specific Reactivity: Antibodies like 12211-1-AP cross-react with human, mouse, and rat CPOX, while others (e.g., NBP2-87205) are human-specific .
Antibody Types and Hosts
| Antibody | Host | Immunogen | Purification Method | Conjugation Options |
|---|---|---|---|---|
| B-9 (Monoclonal) | Mouse | CPOX protein (human/mouse/rat) | Protein-A affinity chromatography | Agarose, HRP, PE, FITC, Alexa Fluor® |
| PAT36B10AT (Monoclonal) | Mouse | Recombinant human CPOX (aa 111–454) | Protein-A affinity chromatography | Unconjugated |
| NBP2-87205 (Polyclonal) | Rabbit | Synthetic peptide (C-terminal region) | Antigen affinity purification | Unconjugated (BSA-free) |
| 12211-1-AP (Polyclonal) | Rabbit | CPOX fusion protein (Ag2858) | Antigen affinity purification | Unconjugated |
Recommended Dilutions
| Application | Dilution Range | Tested Samples |
|---|---|---|
| Western Blotting | 1:1000–1:4000 (WB) | HepG2, K562, Jurkat, mouse/rat liver |
| Immunohistochemistry | 1:50–1:500 (IHC) | Human liver cancer, normal liver |
| Immunofluorescence | 1:100–1:200 (IF) | Jurkat cells (cytoplasmic localization) |
Role in Hereditary Coproporphyria
Mutations in CPOX reduce enzymatic activity, leading to coproporphyrinogen III accumulation. Antibodies detecting CPOX protein levels are critical for diagnosing this disorder and monitoring therapeutic responses .
Photodynamic Diagnosis in Oncology
CPOX upregulation in malignant brain tumors enhances 5-ALA–induced fluorescence, enabling real-time surgical guidance. Immunohistochemical studies confirm CPOX as a biomarker for tumor aggressiveness .
Mechanistic Insights from Animal Models
Product Specs
Q&A
What experimental applications are validated for CPOX antibodies in heme biosynthesis studies?
CPOX antibodies are primarily used to investigate enzymatic activity in the mitochondrial heme biosynthesis pathway. Validated applications include:
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Western blotting (WB): Detect endogenous CPOX at ~36–50 kDa in human, mouse, and rat tissues . Optimal dilution ranges from 1:1,000 to 1:4,000 depending on sample type .
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Immunohistochemistry (IHC): Localize CPOX in formalin-fixed paraffin-embedded (FFPE) tissues, particularly liver and brain tumors, using antigen retrieval with TE buffer (pH 9.0) or citrate buffer (pH 6.0) .
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Immunofluorescence (IF): Visualize mitochondrial CPOX distribution in cultured cells, often paired with organelle-specific markers .
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Enzyme-linked immunosorbent assay (ELISA): Quantify CPOX levels in erythrocyte lysates using monoclonal antibodies like B-9 (IgG2a κ) .
Key Validation Data:
| Application | Sample Type | Recommended Dilution | Observed MW |
|---|---|---|---|
| WB | HepG2, K-562 cells | 1:1,000–1:4,000 | 36 kDa |
| IHC | Human liver cancer | 1:50–1:500 | N/A |
| IF | Mouse hepatocytes | 1:100–1:200 | 50 kDa |
How should researchers validate CPOX antibody specificity in knockdown/knockout models?
Three orthogonal methods are recommended:
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Genetic knockout controls: Compare signal intensity between wild-type and CPOX-knockout cell lines (e.g., CRISPR-edited HepG2) .
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Peptide blocking assays: Pre-incubate antibody with a 10-fold molar excess of immunogen peptide (e.g., residues 111–454 of human CPOX) . A >90% signal reduction confirms specificity.
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Cross-species reactivity testing: Validate across human, mouse, and rat tissues, as CPOX exhibits 85% sequence homology between these species .
What storage conditions preserve CPOX antibody stability for long-term studies?
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Long-term (>1 month): -20°C in 50% glycerol , avoiding freeze-thaw cycles
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Working aliquots: 20–50 µL volumes reduce repeated exposure to temperature fluctuations
How to resolve contradictory CPOX expression data between qRT-PCR and Western blot?
Contradictions often arise from post-translational modifications or tissue-specific isoform expression:
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Post-translational analysis: Treat lysates with phosphatase (e.g., λ-PPase) to detect phosphorylation-induced mobility shifts .
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Isoform-specific probes: Design primers targeting alternative splice variants (e.g., NM_000097.6 vs NM_001308188.1) paired with antibodies recognizing shared epitopes .
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Protein turnover assessment: Conduct cycloheximide chase experiments to compare mRNA half-life (typically 6–8 hr) with protein degradation rates .
What methodological strategies enhance CPOX detection in low-abundance samples?
How to design experiments correlating CPOX expression with 5-ALA–induced fluorescence in brain tumors?
The PMC-validated workflow provides a robust template:
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Tissue preparation:
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5-μm FFPE sections baked at 98°C for 40 min
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Antigen retrieval with TE buffer (pH 9.0)
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Dual staining protocol:
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Primary antibodies: Rabbit anti-CPOX (1:1,200) + Mouse anti-Ki67 (1:100)
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Secondaries: HRP-anti-rabbit IgG + AP-anti-mouse IgG
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Quantification:
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Count positive cells in ≥3 high-power fields (400x magnification)
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Normalize CPOX% to Ki67 proliferation index
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Critical Statistical Considerations:
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Use Wilcoxon test for non-parametric fluorescence intensity comparisons
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Receiver operating characteristic (ROC) analysis determines optimal CPOX cutoff (AUC >0.85 in glioma studies)
What experimental controls are essential when investigating CPOX mutations in coproporphyria?
Addressing cross-reactivity in phylogenetic studies
CPOX antibodies may recognize orthologs beyond target species:
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Epitope mapping: Compare antibody-binding regions across species using CLUSTAL Omega alignments
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Blocking peptides: Use species-specific immunogen peptides (e.g., human: residues 111–454 vs mouse: 105–442)
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Negative controls: Include non-mammalian samples (e.g., yeast) lacking endogenous CPOX
Optimizing co-immunoprecipitation (Co-IP) for CPOX interaction studies
Product Science Overview
Coproporphyrinogen oxidase (CPOX) is an enzyme that plays a crucial role in the biosynthesis of heme, an essential component of hemoproteins such as hemoglobin. The enzyme is involved in the sixth step of the heme production process, where it catalyzes the oxidative decarboxylation of coproporphyrinogen III to protoporphyrinogen IX . This step is vital for the proper functioning of various biological processes, including oxygen transport and cellular respiration.
CPOX is encoded by the CPOX gene in humans. Mutations in this gene can lead to a reduced production of heme, resulting in medical conditions such as hereditary coproporphyria and harderoporphyria . These conditions are characterized by the accumulation of porphyrin precursors in the body, leading to various symptoms, including abdominal pain, neurological disturbances, and photosensitivity .
Mouse anti-human antibodies are secondary antibodies produced by immunizing mice with human immunoglobulins. These antibodies are used in various research and diagnostic applications to detect, sort, or purify human proteins . They are affinity-purified to ensure high specificity and are often conjugated with labels such as horseradish peroxidase (HRP), alkaline phosphatase (AP), or fluorescent dyes to facilitate detection .
Mouse anti-human antibodies are commonly used in techniques such as Western blotting, ELISA, flow cytometry, and immunohistochemistry . These antibodies provide increased versatility and sensitivity through signal amplification, as multiple secondary antibodies can bind to a single primary antibody .
Combining the knowledge of CPOX and mouse anti-human antibodies, Coproporphyrinogen Oxidase, Mouse Anti Human refers to the use of mouse-derived antibodies to detect human CPOX in various experimental settings. This approach is valuable in research focused on understanding the role of CPOX in heme biosynthesis and its implications in diseases such as hereditary coproporphyria.
By using mouse anti-human antibodies specific to CPOX, researchers can study the expression, localization, and activity of this enzyme in human tissues and cells. This information is crucial for developing therapeutic strategies to manage conditions associated with CPOX deficiencies.
