PNPO Antibody

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:

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:

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.