QPRT Antibody

What is QPRT and why is it important in research?

QPRT (Quinolinate phosphoribosyltransferase) is a rate-limiting enzyme involved in the catabolism of quinolinic acid (QA) and encodes the uronic acid pathway. It plays crucial roles in cell cycle progression and cancer cell metabolism . QPRT catalyzes the production of nicotinic acid mononucleotide (NMN), which promotes the synthesis of nicotinamide adenine dinucleotide (NAD+), a molecule essential for cell survival . Its significance in research stems from its emerging role as a potential biomarker in various cancers and its involvement in critical cellular pathways.

How does QPRT function in cellular metabolism and what pathways does it influence?

QPRT functions as a key enzyme in NAD+ biosynthesis by catalyzing the conversion of quinolinic acid to nicotinic acid mononucleotide. Research indicates that QPRT may significantly impact the PI3K/Akt signaling pathway, particularly in cancer contexts. Studies have shown that phosphorylation and protein levels of P-PI3K and P-Akt were significantly increased in cells with foreign expression of QPRT . Furthermore, enhanced phosphorylation and protein levels of PI3K/Akt downstream protein kinase P-MDM2 have been observed, suggesting that QPRT may promote cancer progression through this pathway . This metabolic function makes QPRT an important target for understanding cellular energy regulation and potential therapeutic interventions.

What criteria should be used when selecting a QPRT antibody for specific applications?

When selecting a QPRT antibody, researchers should consider several criteria based on their intended application. For western blot applications, antibodies validated at dilutions between 1:1000-1:4000 have shown optimal results . For immunofluorescence or immunocytochemistry, dilutions between 1:20-1:200 are typically recommended . Flow cytometry applications generally require approximately 0.80 μg per test . Beyond dilution factors, researchers should carefully evaluate:

• Antibody type (monoclonal vs. polyclonal): Monoclonal antibodies like EPR11941(B) offer high specificity, while polyclonal antibodies may provide stronger signals through recognition of multiple epitopes .

• Validated applications: Confirm the antibody has been validated for your specific application (WB, IF, IHC, FC, IP, etc.) .

• Species reactivity: Most QPRT antibodies show validated reactivity with human samples, with some cross-reactivity to mouse samples .

• Validation methods: Prioritize antibodies validated through knockout/knockdown testing to ensure specificity .

How can researchers validate the specificity of QPRT antibodies in their experimental systems?

Validating QPRT antibody specificity is crucial for generating reliable research data. Several methodological approaches are recommended:

• Knockout/knockdown validation: The gold standard for antibody validation involves comparing antibody signal between wild-type and QPRT knockout samples. For example, wild-type and QPRT knockout samples can be subjected to SDS-PAGE, with GAPDH serving as a loading control .

• Western blot analysis: When performing western blotting, researchers should observe a primary band at approximately 31 kDa. Studies have shown that in follicular thyroid carcinoma (FTC) samples, QPRT antibodies detected the expected 34 kDa band, while no band was observed in follicular thyroid adenoma (FTA) samples .

• Multiple detection methods: Validating findings across different techniques (e.g., western blot, immunohistochemistry, and qRT-PCR) strengthens confidence in antibody specificity .

• Positive and negative tissue controls: Include known positive and negative tissue controls based on established QPRT expression patterns across tissue types to confirm expected staining patterns .

What are the optimal storage conditions and handling procedures for maintaining QPRT antibody efficacy?

To maintain optimal QPRT antibody effectiveness, researchers should adhere to the following storage and handling protocols:

What are the established protocols for QPRT detection using western blotting?

For optimal western blot detection of QPRT, researchers should follow these methodological guidelines:

• Sample preparation: Prepare protein lysates from tissues or cell lines of interest. Studies have successfully used various lysates including HepG2 cells and L02 cells for QPRT detection .

• Protein loading: Load appropriate amounts of protein (typically 20-50 μg per lane) on SDS-PAGE gels. Include positive controls such as HepG2 cell lysate where QPRT expression has been confirmed.

• Antibody dilution: Use QPRT antibodies at dilutions between 1:1000-1:4000 for western blotting . The specific dilution may require optimization based on the particular antibody and sample type.

• Incubation conditions: Incubate with primary antibody at room temperature for 1.5 hours or at 4°C overnight. Secondary antibody incubations typically require 1 hour at room temperature .

• Detection: For QPRT, both chemiluminescent and fluorescent detection methods have been validated. Fluorescent detection using IRDye-conjugated secondary antibodies (800CW for rabbit primaries and 680RD for mouse primaries) allows for multiplex detection with loading controls .

• Expected results: Anticipate a band at approximately 31 kDa, though isoforms at 31-33 kDa, 37 kDa, and 42 kDa have been reported .

How can QPRT be effectively detected using immunofluorescence techniques?

For successful immunofluorescence detection of QPRT, researchers should implement the following protocol:

• Cell preparation: Culture cells (HepG2 cells have been validated as positive controls) on coverslips or in chamber slides to appropriate confluence (typically 70-80%) .

• Fixation and permeabilization: Use either paraformaldehyde (4%) for fixation followed by Triton X-100 (0.1-0.5%) for permeabilization, or methanol fixation which simultaneously permeabilizes.

• Blocking: Block non-specific binding using 1-5% BSA or normal serum in PBS for 30-60 minutes at room temperature.

• Primary antibody incubation: Apply QPRT antibody at dilutions between 1:50-1:500 for immunofluorescence . Incubate for 1-2 hours at room temperature or overnight at 4°C.

• Secondary antibody application: After washing, apply fluorophore-conjugated secondary antibodies appropriate for the host species of the primary antibody.

• Nuclear counterstaining: Include DAPI or Hoechst stain to visualize nuclei, which aids in cellular localization assessment of QPRT.

• Mounting and imaging: Mount slides using anti-fade mounting medium and image using appropriate fluorescence microscopy techniques.

• Controls: Include negative controls (primary antibody omission) and positive controls (cells known to express QPRT, such as HepG2) .

What are the methodological considerations for flow cytometric analysis of QPRT?

Flow cytometric detection of QPRT requires intracellular staining techniques due to its cytoplasmic localization. The following protocol outline provides methodological guidance:

• Cell preparation: Harvest cells (e.g., HepG2 cells as positive controls) and wash in PBS containing 2% FBS .

• Fixation and permeabilization: Fix cells with 4% paraformaldehyde for 10-15 minutes at room temperature, then permeabilize using 0.1% saponin or a commercial permeabilization buffer.

• Blocking: Block non-specific binding with 2-5% normal serum from the same species as the secondary antibody.

• Primary antibody staining: Incubate cells with QPRT antibody, using approximately 0.80 μg per test for intracellular flow cytometry .

• Secondary antibody application: If using an unconjugated primary antibody, wash cells and incubate with fluorophore-conjugated secondary antibody appropriate for the flow cytometer's laser configuration.

• Controls: Include isotype controls, unstained cells, and single-color controls for compensation if performing multicolor flow cytometry.

• Data analysis: When analyzing flow cytometry data, examine the shift in fluorescence intensity compared to control samples to determine QPRT expression levels.

• Gating strategy: Implement appropriate gating strategies to exclude cell debris, doublets, and dead cells before analyzing QPRT expression.

How is QPRT antibody used in thyroid cancer diagnosis and what are the sensitivity/specificity considerations?

QPRT antibody has emerged as a potentially valuable diagnostic tool for differentiating follicular thyroid carcinoma (FTC) from follicular thyroid adenoma (FTA), a challenging distinction in thyroid pathology. Research findings provide the following methodological insights:

• Diagnostic performance: In retrospective studies, QPRT immunohistochemistry achieved 72% diagnostic accuracy (107 out of 149 cases) in distinguishing FTC from FTA . The sensitivity was 65% (47/72 positive FTCs) and specificity was 73% (60/77 negative FTAs) .

• Staining patterns: Positive QPRT immunostaining was observed in 65% of follicular thyroid carcinomas, including minimal invasive variants, while only 22% of follicular adenomas showed positivity .

• Subtype considerations: The sensitivity varied among FTC subtypes, with 60% sensitivity in minimal invasive FTC and 75% in widely invasive FTC . In prospective studies, 85.7% of minimal invasive FTCs showed positive QPRT staining .

• Special cases: Oxyphilic FTA showed higher positivity rates (66.7%, 6 out of 9 cases), suggesting potential limitations in this subtype . Additionally, 27.4% of nodular goiter cases showed focal positivity, often in areas with oxyphilic metaplasia .

• Western blot validation: Western blotting confirmed antibody specificity, showing bands at 34 kD in FTC samples but not in FTA samples .

What is the role of QPRT in breast cancer progression and how do antibodies help elucidate these mechanisms?

QPRT has been identified as an independent prognostic factor in invasive breast cancer, with antibody-based techniques revealing several key mechanistic insights:

• Prognostic significance: QPRT expression has been associated with breast cancer prognosis, with studies suggesting it may function as an independent prognostic factor .

• Cellular effects: Research utilizing QPRT antibodies has demonstrated that QPRT promotes growth, migration, and invasion of breast cancer cells while inhibiting apoptosis .

• Signaling pathway activation: Western blotting with QPRT and pathway-specific antibodies has revealed that QPRT activates the PI3K/Akt signaling pathway in breast cancer cells . Phosphorylation and protein levels of P-PI3K and P-Akt were significantly increased in cells overexpressing QPRT .

• Downstream effects: QPRT expression enhanced phosphorylation and protein levels of the PI3K/Akt downstream protein kinase P-MDM2, further supporting its role in this signaling cascade .

• Metabolic influence: QPRT's catalytic function in producing nicotinic acid mononucleotide (NMN) promotes NAD+ synthesis, which is crucial for cell survival and may contribute to cancer cell metabolic reprogramming .

• Expression analysis: Pan-cancer analysis using QPRT antibodies has revealed significant upregulation of QPRT expression in multiple tumor types, including breast cancer, colorectal cancer, and glioblastoma multiforme .

How can researchers quantitatively assess QPRT expression levels in clinical samples?

Several quantitative methods using QPRT antibodies can be employed to assess expression levels in clinical samples:

• ELISA quantification: Human QPRT ELISA kits provide a sandwich (quantitative) method for measuring QPRT in various human sample types including cerebral spinal fluid, saliva, urine, plasma, cell culture supernatant, milk, and serum . This approach offers quantitative concentration measurements with coefficient of variation (C.V.) values of approximately 5.5-10.1% .

• Immunohistochemical scoring: Semi-quantitative assessment of QPRT expression in tissue sections can be performed using scoring systems that evaluate staining intensity and percentage of positive cells . This approach has been successfully applied in thyroid cancer studies, where positive/negative QPRT status showed correlations with clinical outcomes.

• Western blot densitometry: Quantitative analysis of western blot bands can be performed using densitometry software to compare QPRT expression levels between samples, normalizing to loading controls such as GAPDH .

• Flow cytometry: Intracellular flow cytometry provides quantitative assessment of QPRT expression at the single-cell level, allowing for population analysis and potential correlation with other cellular markers .

• qRT-PCR correlation: Although antibody-independent, qRT-PCR measurements of QPRT mRNA can be correlated with protein expression data from antibody-based methods to strengthen quantitative findings .

What are common technical challenges when working with QPRT antibodies and how can they be addressed?

Researchers working with QPRT antibodies may encounter several technical challenges that can be addressed with appropriate troubleshooting strategies:

• Non-specific bands in western blotting:

  • Challenge: Detection of multiple bands beyond the expected 31 kDa QPRT band.

  • Solution: Validate antibody specificity using QPRT knockout samples as negative controls . Optimize antibody dilution (1:1000-1:4000 for western blotting) . Consider using monoclonal antibodies like EPR11941(B) which may offer higher specificity .

• Variable staining patterns in immunohistochemistry:

  • Challenge: Inconsistent staining results, particularly in oxyphilic areas.

  • Solution: Note that oxyphilic FTA shows higher positivity rates (66.7%) . Include appropriate positive and negative tissue controls. Standardize fixation protocols, as fixation time can affect epitope accessibility.

• Low signal-to-noise ratio:

  • Challenge: High background with weak specific QPRT signal.

  • Solution: Optimize blocking conditions using 5% BSA or normal serum. Increase primary antibody incubation time (overnight at 4°C rather than shorter incubations at room temperature). Ensure adequate washing steps between antibody applications.

• Cross-reactivity concerns:

  • Challenge: Potential cross-reactivity with other proteins.

  • Solution: Use antibodies that have undergone affinity purification . Validate specificity through knockout/knockdown experiments or peptide competition assays .

• Reproducibility issues:

  • Challenge: Inconsistent results between experiments.

  • Solution: Standardize protocols including sample preparation, antibody dilutions, and incubation times. Maintain proper antibody storage conditions (-20°C, avoiding frequent freeze-thaw cycles) .

How can researchers integrate QPRT antibody-based findings with other molecular techniques for comprehensive pathway analysis?

For comprehensive pathway analysis involving QPRT, researchers should consider integrating antibody-based findings with complementary molecular techniques:

• Multi-omics approach:

  • Combine QPRT protein expression data from antibody-based methods with transcriptomic data (RNA-seq or microarray) to correlate protein and mRNA levels.

  • Integrate with metabolomic analysis focusing on NAD+ metabolism components, given QPRT's role in nicotinic acid mononucleotide production .

• Pathway validation strategies:

  • When investigating QPRT's role in the PI3K/Akt pathway, combine western blotting data of QPRT with phosphorylation status of pathway components (P-PI3K, P-Akt, P-MDM2) .

  • Implement pathway inhibitor studies (e.g., PI3K inhibitors) to confirm QPRT's functional impact on signaling.

• Functional validation:

  • Correlate QPRT antibody detection with functional assays such as cell proliferation, migration, invasion, and apoptosis assays .

  • Utilize QPRT overexpression and knockdown/knockout approaches followed by antibody-based detection of pathway components to establish causality.

• Advanced imaging techniques:

  • Combine immunofluorescence detection of QPRT with co-localization studies using antibodies against interacting proteins or pathway components.

  • Consider proximity ligation assays to detect protein-protein interactions involving QPRT.

• Clinical correlation:

  • Integrate immunohistochemical QPRT detection in patient samples with clinical outcome data to establish prognostic relevance .

  • Perform gene set enrichment analysis (GSEA) using QPRT expression data to identify associated pathways and biological processes .

What emerging research directions are utilizing QPRT antibodies beyond current applications?

Several emerging research directions are expanding the utility of QPRT antibodies beyond conventional applications:

• Single-cell analysis:

  • Application of QPRT antibodies in single-cell proteomics to understand heterogeneity of expression within tumor microenvironments.

  • Integration with single-cell RNA sequencing data to correlate protein and transcript levels at the individual cell level.

• Therapeutic targeting validation:

  • Use of QPRT antibodies to evaluate the efficacy of potential therapeutic approaches targeting QPRT or downstream pathways.

  • Development of antibody-drug conjugates targeting QPRT in cancer cells with high expression.

• Liquid biopsy development:

  • Investigation of secreted or released QPRT in body fluids as potential biomarkers using ELISA techniques .

  • Correlation of circulating QPRT levels with tissue expression and disease progression.

• Tissue microarray (TMA) studies:

  • Large-scale application of QPRT antibodies in TMAs to establish expression patterns across diverse cancer types and subtypes.

  • Correlation with other biomarkers to develop comprehensive prognostic panels.

• Post-translational modification analysis:

  • Development of modification-specific QPRT antibodies (phospho-QPRT, acetyl-QPRT) to understand regulatory mechanisms affecting QPRT function.

  • Investigation of how these modifications might alter QPRT's catalytic activity or interaction with other proteins.

• Combination biomarker approaches:

  • Integration of QPRT with other biomarkers for improved diagnostic accuracy in challenging cases, such as the FTA/FTC distinction where QPRT alone shows 72% accuracy .

  • Development of multiplexed immunofluorescence panels including QPRT for comprehensive tumor profiling.

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