PNPO Antibody

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Description

Introduction and Definition

Pyridoxine 5′-phosphate oxidase (PNPO) is an enzyme critical for converting pyridoxine 5′-phosphate and pyridoxamine 5′-phosphate into pyridoxal 5′-phosphate (PLP), the active form of vitamin B6 . PNPO Antibody refers to immunoglobulins designed to detect and study PNPO protein expression in biological samples. These antibodies are indispensable in research for elucidating PNPO’s role in cancer progression, metabolic regulation, and therapeutic targeting .

Applications and Reactivity

PNPO antibodies are validated for diverse techniques, including:

ApplicationDescription
Western Blot (WB)Detects PNPO protein expression in lysates, confirming knockdown efficacy .
Immunohistochemistry (IHC)Identifies PNPO localization in tumor tissues (e.g., ovarian, breast cancers) .
Immunofluorescence (IF/ICC)Visualizes subcellular distribution of PNPO in cultured cells .
Enzyme-Linked Immunosorbent Assay (ELISA)Quantifies PNPO levels in serum or lysates .

Species Reactivity:

  • Human, mouse, rat: Broadly applicable across mammalian models .

  • Cited Reactivity: Human and mouse focus in most studies .

Overexpression in Cancers

PNPO is upregulated in ovarian, breast, and pan-cancer contexts, correlating with aggressive phenotypes .

Cancer TypeEffect of PNPO KnockdownKey FindingsReferences
Epithelial OvarianInduces apoptosis, G2/M arrest, reduced invasionTumor volume ↓50% in xenograft models; PLP feedback inhibits PNPO expression .
Breast (IDC)Decreased proliferation, migration, colony formationPNPO serum levels correlate with metastasis; linked to PR expression .
Pan-CancerPredicts prognosis (ESCA, KIRC, PRAD, OV, UVM)Amplified PNPO linked to drug sensitivity and immune infiltration .

Mechanistic Insights

  • TGF-β Signaling: Downregulates PNPO via miR-143-3p upregulation .

  • PLP Feedback Loop: Exogenous PLP reduces PNPO expression, modulating vitamin B6 metabolism .

Product Specs

Buffer
Preservative: 0.03% Proclin 300
Composition: 50% Glycerol, 0.01M PBS, pH 7.4
Form
Liquid
Lead Time
Generally, we can ship the products within 1-3 business days after receiving your order. Delivery time may vary depending on the purchase method or location. Please consult your local distributor for specific delivery times.
Synonyms
EC 1.4.3.5 antibody; FLJ10535 antibody; PDXPO antibody; PNPO antibody; PNPO_HUMAN antibody; Pyridoxal 5' phosphate synthase antibody; pyridoxamine 5' phosphate oxidase antibody; Pyridoxamine phosphate oxidase antibody; Pyridoxamine-phosphate oxidase antibody; pyridoxine 5' phosphate oxidase antibody; Pyridoxine-5'-phosphate oxidase antibody
Target Names
PNPO
Uniprot No.

Target Background

Function
This antibody catalyzes the oxidation of either pyridoxine 5'-phosphate (PNP) or pyridoxamine 5'-phosphate (PMP) into pyridoxal 5'-phosphate (PLP).
Gene References Into Functions
  1. TGF-beta1-mediated PNPO expression was at least in part through the upregulation of miR-143-3p in epithelial ovarian cancer. PMID: 29238081
  2. Data supports a pathogenic role of the c.347G>A (p.Arg116Gln) mutation in PNPO deficiency; the later onset of symptoms and the milder epilepsy phenotype of these expand the disease phenotype PMID: 28818555
  3. Exome sequencing revealed that the patient was compound heterozygous for pathogenic mutations [c.546+1G>A (IVS5+1 G>A) and c.620delG (p.G207VfsX215)] in the PNPO gene. PMID: 26535729
  4. One sequence variant, R116Q, a single nucleotide polymorphism that has been reported in the general population, was found to have an effect on PNPO activity. PMID: 24645144
  5. This research challenges the paradigm of exclusive PLP responsiveness in patients with pyridoxal 5'-phosphate oxidase deficiency and highlights the importance of consecutive testing of pyridoxine and PLP in neonates with antiepileptic drug-resistant seizures. PMID: 24658933
  6. Generalized epilepsies implicate susceptibility genes: CHRM3 at 1q43, VRK2 at 2p16.1, ZEB2 at 2q22.3, SCN1A at 2q24.3 and PNPO at 17q21.32. PMID: 22949513
  7. This study provides evidence that a single arginine residue of the C terminus of pyridoxal 5'-phosphate synthase is responsible for coordinating co-operativity in this elaborate protein machinery PMID: 21283685
  8. This research describes a combined computer and biochemical approach to characterize human pyridoxine 5'-phosphate oxidase (PNPO). PMID: 15182361
  9. Several mutations appear to cause epilepsy PMID: 15772097
  10. This study examined 8 single nucleotide polymorphisms (SNPs) in PNPO and its 5'-flanking regions in 359 schizophrenia patients and 582 control subjects. PMID: 17851041
  11. Sequencing of the PNPO gene revealed a novel homozygous nonsense mutationt in exon 3 in the neonatal and infantile seizure. PMID: 18485777
  12. The PNP oxidase R229W mutation identified in patients with neonatal epileptic encephalopathy significantly affects the catalytic efficiency of the enzyme. PMID: 19759001

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Database Links

HGNC: 30260

OMIM: 603287

KEGG: hsa:55163

STRING: 9606.ENSP00000225573

UniGene: Hs.631742

Involvement In Disease
Pyridoxine-5'-phosphate oxidase deficiency (PNPOD)
Protein Families
Pyridoxamine 5'-phosphate oxidase family

Q&A

What is PNPO and what is its biological function?

PNPO (pyridoxamine 5'-phosphate oxidase) is an enzymic protein crucial for the synthesis of pyridoxal 5'-phosphate (PLP), the active form of vitamin B6. It has a calculated molecular weight of approximately 30 kDa . Initially, PNPO was primarily studied in relation to epilepsy, but recent research has revealed its involvement in tumorigenesis in ovarian and breast cancers, partially regulated through the TGF-β signaling pathway . PNPO has been implicated in various cellular processes including autophagy, transport systems, and oxidative stress responses, making it an important target for both neurological and cancer research .

What types of PNPO antibodies are available for research applications?

Researchers can access different types of PNPO antibodies for experimental work:

  • Based on host/antibody class:

    • Polyclonal antibodies (e.g., rabbit IgG)

    • Monoclonal antibodies (e.g., mouse IgG)

  • Based on conjugation:

    • Unconjugated antibodies for standard applications

    • Fluorescent-conjugated antibodies (e.g., DyLight 405) for direct visualization

  • Based on immunogen:

    • Antibodies raised against full-length recombinant PNPO protein

    • Antibodies raised against PNPO fusion proteins

The choice between these types depends on the specific application and experimental design requirements.

In which species has PNPO expression been documented and which antibodies show cross-reactivity?

PNPO expression has been documented in multiple species, and antibodies show varying cross-reactivity:

AntibodyTested ReactivityCited Reactivity
15552-1-AP (Polyclonal)Human, mouse, ratHuman, mouse
OTI1G9 (Monoclonal)Human, mouse, ratNot specified

These antibodies have been validated in various tissue types including human brain, heart, and liver tissues, as well as mouse and rat brain tissues . This cross-reactivity information is essential when designing experiments involving different model organisms.

What are the recommended dilutions for PNPO antibodies in different applications?

Proper antibody dilution is critical for successful experiments. Based on validated protocols, here are the recommended dilutions for PNPO antibody 15552-1-AP:

ApplicationDilution
Western Blot (WB)1:500-1:3000
Immunoprecipitation (IP)0.5-4.0 μg for 1.0-3.0 mg of total protein lysate
Immunohistochemistry (IHC)1:20-1:200
Immunofluorescence (IF)/ICC1:10-1:100

It is strongly recommended that researchers titrate these antibodies in their specific testing systems to obtain optimal results, as sample-dependent variations may occur . For monoclonal antibodies like OTI1G9, optimal dilutions should be experimentally determined for each application .

What antigen retrieval methods are recommended for PNPO immunohistochemistry?

For optimal PNPO detection in immunohistochemistry on paraffin-embedded tissues, the following antigen retrieval methods are recommended:

  • Primary recommendation: TE buffer at pH 9.0

  • Alternative method: Citrate buffer at pH 6.0

These recommendations are based on validated protocols for human liver tissue . In published studies, researchers have successfully used PNPO antibody at 1:400 dilution following antigen retrieval and blocking with 10% normal goat serum for 40 minutes at room temperature . The protocol typically involves overnight incubation with the primary antibody at 4°C, followed by appropriate secondary antibody incubation at room temperature for 1 hour.

How can I validate PNPO antibody specificity in my experimental system?

Validating antibody specificity is essential for reliable results. Consider these methodological approaches:

  • Positive and negative tissue controls: Use tissues known to express or lack PNPO. Validated positive samples include MCF-7 cells, human brain, heart, and liver tissues, and mouse/rat brain tissues .

  • siRNA knockdown validation: Treat cells with PNPO-specific siRNA and confirm reduction in signal compared to scrambled siRNA controls. This approach has been successfully used in ovarian cancer cell lines .

  • Overexpression studies: Compare signal in cells transfected with PNPO expression vectors versus empty vector controls.

  • Multiple antibody validation: Use antibodies from different sources or those recognizing different epitopes to confirm consistent detection patterns.

  • Western blot verification: Confirm detection of a band at the expected molecular weight (30 kDa for PNPO) .

How does PNPO expression relate to autophagy and lysosomal function in cancer cells?

Research has revealed sophisticated relationships between PNPO and autophagy/lysosomal processes:

  • Enhanced lysosomal biogenesis: PNPO overexpression enhances the biogenesis and perinuclear distribution of lysosomes in ovarian cancer cells .

  • Autophagosome degradation: Elevated PNPO levels promote the degradation of autophagosomes and boost autophagic flux .

  • PNPO-LAMP2 axis: The autolysosome marker LAMP2 is upregulated in PNPO-overexpressing ovarian cancer cells. Silencing LAMP2 suppresses cell growth, induces apoptosis, and blocks PNPO action, suggesting the existence of a functional PNPO-LAMP2 axis .

  • Therapeutic implications: Chloroquine has been shown to counteract PNPO's promotion effect on autophagic flux and inhibit ovarian cancer cell survival, facilitating the inhibitory effect of PNPO-shRNA on tumor growth in vivo .

These findings suggest that PNPO antibodies can be valuable tools for investigating autophagy regulation in cancer research, particularly when used in combination with autophagy markers like LC3A/B and LAMP2.

What is the role of PNPO in chemoresistance, and how can antibodies be used to study this phenomenon?

PNPO has emerged as a potentially important factor in chemoresistance:

  • Overexpression in resistant cells: PNPO is overexpressed in paclitaxel-resistant ovarian cancer cells, suggesting its involvement in resistance mechanisms .

  • Sensitization effect: PNPO-siRNA has been shown to enhance paclitaxel sensitivity both in vitro and in vivo, indicating its potential as a target for overcoming resistance .

  • Methodological approach: To study this phenomenon, researchers can use PNPO antibodies to:

    • Compare PNPO expression levels between chemosensitive and chemoresistant cell lines via Western blot or IHC

    • Monitor changes in PNPO expression during acquired resistance development

    • Evaluate PNPO localization changes in resistant cells using immunofluorescence

    • Assess the correlation between PNPO expression and patient response to chemotherapy in clinical samples

  • Combinatorial analysis: Combine PNPO antibodies with markers of autophagic flux, cell cycle regulators (e.g., cyclin B1, phosphorylated CDK1), and cell death markers to comprehensively characterize the molecular mechanisms of PNPO-mediated chemoresistance.

How can I reduce background when using PNPO antibodies on tissues from the same species as the antibody host?

When using mouse-derived antibodies (like OTI1G9) on mouse tissues, researchers often encounter high background signal due to endogenous mouse immunoglobulins. To overcome this challenge:

  • Mouse-On-Mouse blocking reagents: Use specialized blocking reagents designed to minimize background in mouse-on-mouse applications. Commercial options include those available under catalog numbers PK-2200-NB and MP-2400-NB .

  • Isotype controls: Include appropriate isotype controls to distinguish specific staining from background.

  • Antibody fragmentation: Consider using F(ab) or F(ab')2 fragments instead of whole IgG antibodies.

  • Alternative detection systems: Use detection systems specifically designed for mouse-on-mouse applications, such as polymer-based systems that minimize cross-reactivity.

  • Species-specific secondary antibodies: Ensure secondary antibodies are highly cross-adsorbed against the species being examined.

What are the optimal storage conditions for maintaining PNPO antibody activity?

Proper storage is crucial for antibody stability and reproducible results:

  • Temperature: Store PNPO antibodies at -20°C for long-term storage. They are typically stable for one year after shipment under these conditions .

  • Formulation considerations:

    • Most PNPO antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

    • Some conjugated antibodies (like DyLight 405-conjugated PNPO antibodies) should be stored at 4°C in the dark

  • Aliquoting: While aliquoting is generally recommended for antibodies, for certain formulations (like those with 50% glycerol), it may be unnecessary for -20°C storage .

  • Freeze-thaw cycles: Minimize freeze-thaw cycles regardless of formulation to maintain antibody performance.

  • Special considerations: Some antibody preparations (20μl sizes) contain 0.1% BSA, which may affect certain applications .

How can I quantify PNPO expression in immunohistochemistry specimens?

For reliable quantification of PNPO expression in IHC specimens:

  • Digital image analysis: Use software tools like ImageJ with the IHC Profiler plugin for automated quantitative evaluation, as demonstrated in published PNPO research .

  • Standardized scoring systems: Implement H-score, Allred score, or similar standardized systems that account for both staining intensity and percentage of positive cells.

  • Multi-region analysis: Analyze multiple fields per sample (minimum 3-5) to account for heterogeneity.

  • Control normalization: Include positive and negative controls in each batch for normalization and comparison.

  • Blinded assessment: Have specimens scored by at least two independent observers blinded to experimental conditions to minimize bias.

  • Correlation with other methods: Validate IHC findings with orthogonal methods like Western blot or qPCR when possible.

How should I interpret PNPO expression changes in relation to disease biomarkers?

Interpreting PNPO expression in a disease context requires consideration of multiple factors:

  • Prognostic significance: PNPO has been identified as a tissue biomarker with prognostic implications in ovarian cancer. Higher expression levels may correlate with poorer patient outcomes .

  • Correlation with cell cycle markers: PNPO expression significantly impacts cell proliferation by regulating cyclin B1 and phosphorylated CDK1, affecting the G2M phase of the cell cycle . When analyzing PNPO, consider co-staining or parallel analysis of these cell cycle regulators.

  • Autophagy pathway correlation: Evaluate PNPO expression in relation to autophagy markers like LAMP2 and LC3A/B. The PNPO-LAMP2 axis appears particularly important in cancer progression .

  • Metabolic implications: In PNPO deficiency contexts, correlation with metabolic markers is informative. Elevated glycine and reduced arginine concentrations in both CSF and plasma, along with slightly elevated plasma threonine, have been associated with PNPO deficiency .

  • Statistical analysis: Use appropriate statistical methods to establish significant correlations between PNPO expression and other biomarkers or clinical parameters.

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