QPRT Antibody

What is QPRT and why is it relevant to cancer research?

QPRT (Quinolinate phosphoribosyltransferase) is a rate-limiting enzyme that encodes the uronic acid pathway, playing crucial roles in cell cycle progression and cancer cell metabolism. The enzyme catalyzes the production of nicotinic acid mononucleotide (NMN), which subsequently promotes the synthesis of nicotinamide adenine dinucleotide (NAD+), a molecule critical for cell survival . QPRT has demonstrated progrowth effects on breast cancer tumor cells and has been associated with tumor progression in multiple cancer types. Pan-cancer analysis has revealed that QPRT is significantly expressed in 16 different tumor types including breast cancer (BRCA), colon adenocarcinoma (COAD), and glioblastoma multiforme (GBM) . Its expression appears to be particularly relevant in HER2+ breast cancer, where it serves as an independent prognostic factor .

What are the key considerations when selecting a QPRT antibody for research?

When selecting a QPRT antibody, researchers should consider:

• Specificity: Ensure the antibody specifically recognizes QPRT with minimal cross-reactivity to other proteins. The antibody should be validated through techniques such as western blotting in both overexpression systems and knockdown/knockout controls.

• Application compatibility: Confirm the antibody has been validated for your specific application (WB, IHC, IF/ICC, ELISA, etc.). For example, antibody 25174-1-AP has been validated for western blot (1:1000-1:4000 dilution) and immunofluorescence (1:20-1:200 dilution) .

• Species reactivity: Verify the antibody recognizes QPRT in your experimental model species. The 25174-1-AP antibody has confirmed reactivity with human samples and predicted reactivity with other species .

• Isoform recognition: QPRT exists in multiple isoforms with molecular weights ranging from 31-33 kDa, 37 kDa, and 42 kDa. Ensure the selected antibody recognizes the isoform relevant to your research .

• Conjugation status: Determine whether your application requires an unconjugated antibody (like 25174-1-AP) or a fluorescently conjugated version (like CL488-25174 conjugated to CoraLite® Plus 488) .

What are the typical applications for QPRT antibodies in research?

QPRT antibodies can be utilized across multiple research applications:

• Western Blotting (WB): Used to detect and quantify QPRT protein expression levels in cell or tissue lysates. The recommended dilution for antibody 25174-1-AP is 1:1000-1:4000 .

• Immunofluorescence (IF)/Immunocytochemistry (ICC): Employed to visualize the subcellular localization of QPRT within cells. Immunohistochemical results have shown that QPRT localizes in the cytoplasm, cell membrane, and nucleus of breast cancer cells . The recommended dilution for antibody 25174-1-AP is 1:20-1:200, while for the fluorescent-conjugated CL488-25174, it is 1:50-1:500 .

• Flow Cytometry: Used to quantify QPRT expression at the single-cell level, enabling analysis of expression heterogeneity within cell populations. For the CL488-25174 antibody, the recommended concentration is 0.80 μg per 10^6 cells in a 100 μl suspension .

• Immunohistochemistry (IHC): Utilized to examine QPRT expression in tissue sections, which is particularly valuable for analyzing patient samples and comparing expression between normal and cancerous tissues .

How should QPRT antibody validation be performed for breast cancer research?

A robust QPRT antibody validation strategy for breast cancer research should include:

• Positive and negative controls: Use cell lines with known QPRT expression (such as HepG2 cells as positive controls) alongside QPRT knockdown or knockout models as negative controls .

• Multiple detection methods: Validate the antibody using complementary techniques such as western blotting, immunofluorescence, and flow cytometry to ensure consistent results across platforms.

• Breast cancer subtype specificity: Since QPRT expression varies significantly across breast cancer subtypes, with highest expression in HER2+ breast cancer, validate the antibody across multiple breast cancer cell lines representing different molecular subtypes (luminal, HER2+, triple-negative) .

• Antibody titration: Perform careful titration experiments within the recommended dilution ranges (e.g., 1:1000-1:4000 for WB) to determine optimal concentrations for specific sample types and experimental conditions .

• Evaluation of subcellular localization: Confirm that the antibody appropriately detects QPRT in its reported subcellular locations (cytoplasm, cell membrane, and nucleus) in breast cancer cells through immunofluorescence or immunohistochemistry .

What are the optimal protocols for detecting QPRT in breast cancer tissue samples?

For optimal detection of QPRT in breast cancer tissue samples, researchers should consider:

• Tissue fixation and processing: Use 10% neutral-buffered formalin fixation followed by paraffin embedding for consistent results. Overfixation can mask epitopes and reduce antibody binding.

• Antigen retrieval methods: Heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) can improve QPRT detection. Optimize the retrieval method based on preliminary experiments.

• Blocking and antibody incubation: Implement thorough blocking with serum-free protein block to minimize non-specific binding. For primary antibody incubation, use the recommended dilution (for IHC) and incubate overnight at 4°C for optimal sensitivity.

• Detection system selection: Use a highly sensitive detection system like polymer-based HRP systems for chromogenic detection or fluorescent-conjugated secondary antibodies for fluorescence detection.

• Comparison with normal tissue controls: Always include normal breast tissue controls for comparison, as QPRT shows positive expression in pathological breast cancer tissues but not in normal tissues .

• Digital quantification: For prognostic studies, implement digital image analysis to quantify QPRT expression objectively, especially when evaluating its potential as an independent prognostic factor .

How can QPRT protein isoform specificity be determined experimentally?

To determine QPRT protein isoform specificity experimentally:

• SDS-PAGE resolution: Use gradient gels (4-20%) to effectively separate the different QPRT isoforms (31-33 kDa, 37 kDa, and 42 kDa) .

• Isoform-specific positive controls: Generate or obtain expression constructs for specific QPRT isoforms to serve as definitive positive controls in western blotting experiments.

• Mass spectrometry validation: Perform immunoprecipitation using the QPRT antibody followed by mass spectrometry analysis to identify which specific isoforms are being detected.

• Multiple antibody comparison: Use antibodies targeting different epitopes of QPRT to confirm isoform detection patterns.

• Tissue/cell type expression profiling: Different tissues or cell types may preferentially express certain QPRT isoforms. Profile expression across various breast cancer subtypes to determine if isoform expression correlates with specific pathological features.

How can QPRT antibodies be used to investigate the PI3K/Akt signaling pathway in breast cancer?

QPRT antibodies can be instrumental in elucidating the relationship between QPRT and the PI3K/Akt signaling pathway in breast cancer:

• Co-immunoprecipitation experiments: Use QPRT antibodies to immunoprecipitate QPRT and associated proteins, followed by western blotting for PI3K/Akt pathway components to identify direct protein-protein interactions.

• Proximity ligation assays: Implement this technique using QPRT antibodies in combination with antibodies against PI3K/Akt pathway components to visualize and quantify potential interactions at the single-molecule level within cells.

• Sequential western blotting: After manipulating QPRT expression (overexpression or knockdown), use QPRT antibodies alongside phospho-specific antibodies for PI3K (P-PI3K), Akt (P-Akt), and downstream effectors like MDM2 (P-MDM2) to assess pathway activation, as demonstrated in previous research where QPRT overexpression significantly increased phosphorylation of these proteins .

• Immunofluorescence co-localization: Perform dual-labeling experiments with QPRT antibodies and antibodies against PI3K/Akt pathway components to assess potential co-localization at subcellular levels.

• Inhibitor studies: Use specific PI3K/Akt pathway inhibitors in combination with QPRT antibody-based detection methods to determine whether QPRT-mediated effects are dependent on PI3K/Akt signaling.

• In vivo tumor models: Utilize QPRT antibodies for immunohistochemical analysis of xenograft tumors with manipulated QPRT expression to correlate QPRT levels with PI3K/Akt pathway activation and tumor progression in vivo .

How can QPRT expression be evaluated as a prognostic biomarker in different breast cancer subtypes?

To evaluate QPRT as a prognostic biomarker across breast cancer subtypes:

What methodological approaches can resolve contradictory findings regarding QPRT expression and function?

To address contradictory findings regarding QPRT expression and function, researchers should consider:

• Antibody standardization: Use the same well-validated QPRT antibody across studies, with clearly reported catalog numbers, dilutions, and detection methods.

• Context-specific analysis: Recognize that QPRT functions may be context-dependent, varying across tissue types, cancer subtypes, and experimental models. Compare results only within similar experimental contexts.

• Comprehensive isoform analysis: Since QPRT has multiple isoforms (31-33 kDa, 37 kDa, and 42 kDa), inconsistent findings might result from detection of different isoforms . Use antibodies and techniques that can discriminate between isoforms.

• Subcellular localization considerations: Account for QPRT's presence in multiple cellular compartments (cytoplasm, membrane, nucleus) when interpreting functional results, as localization may influence function .

• Pathway integration analysis: Evaluate QPRT within the broader context of connected pathways (e.g., NAD+ synthesis, PI3K/Akt signaling) rather than in isolation, using systems biology approaches.

• Meta-analysis of expression data: Integrate findings from multiple studies and databases (e.g., TCGA, GTEx, GEO) using consistent bioinformatic pipelines to identify robust patterns beyond individual study limitations.

• Functional validation through multiple approaches: Combine genetic (CRISPR/Cas9, RNAi) and pharmacological approaches targeting QPRT, validating findings with rescue experiments and multiple complementary assays.

How should researchers interpret discrepancies between QPRT mRNA and protein expression in cancer studies?

When faced with discrepancies between QPRT mRNA and protein expression:

• Post-transcriptional regulation analysis: Investigate potential microRNA-mediated regulation of QPRT mRNA by performing correlation analyses between QPRT protein levels (measured with antibodies) and expression of predicted regulatory microRNAs.

• Protein stability assessment: Evaluate QPRT protein stability using cycloheximide chase experiments and QPRT antibody detection to determine if differences in protein half-life, rather than transcription, explain expression variations.

• Translation efficiency evaluation: Use polysome profiling followed by RT-qPCR to assess translation efficiency of QPRT mRNA, which may not correlate directly with mRNA abundance.

• Technical validation: Confirm both mRNA measurements (using multiple primer sets targeting different transcript regions) and protein measurements (using different antibodies and detection methods) to rule out technical artifacts.

• Compartmentalized expression analysis: Assess whether QPRT protein is sequestered in particular cellular compartments that may affect extraction efficiency and apparent expression levels, using subcellular fractionation followed by western blotting.

• Consideration of isoform-specific expression: Determine whether specific QPRT isoforms are differentially regulated at the transcriptional versus post-transcriptional level, potentially explaining discrepancies in total mRNA versus protein measurements.

What are common sources of false positives/negatives when using QPRT antibodies, and how can they be mitigated?

Common sources of false results and mitigation strategies include:

• Antibody cross-reactivity:

  • False positive: Antibody may recognize proteins with similar epitopes to QPRT

  • Mitigation: Validate specificity using QPRT knockout/knockdown controls; perform peptide competition assays

• Inadequate antigen retrieval:

  • False negative: Insufficient retrieval of QPRT epitopes in fixed tissues

  • Mitigation: Optimize antigen retrieval conditions (method, buffer, duration, temperature)

• Inappropriate antibody dilution:

  • False negative: Too dilute antibody fails to detect QPRT

  • False positive: Too concentrated antibody causes background staining

  • Mitigation: Perform careful titration experiments within the recommended ranges (e.g., 1:1000-1:4000 for WB, 1:20-1:200 for IF/ICC)

• Detection system limitations:

  • False negative: Insensitive detection system fails to detect low QPRT expression

  • Mitigation: Use amplification systems (e.g., TSA) for low-abundance targets

• Tissue fixation variables:

  • False negative: Overfixation masking epitopes

  • Mitigation: Standardize fixation protocols; optimize antibody for specific fixation methods

• Sample degradation:

  • False negative: Protein degradation before fixation/extraction

  • Mitigation: Minimize time between sample collection and processing; use protease inhibitors

• Isoform specificity issues:

  • False negative: Antibody may not recognize all QPRT isoforms

  • Mitigation: Use antibodies validated to detect multiple isoforms or specific isoforms of interest

How can QPRT antibodies be effectively used in multiplexed assays to study QPRT in the tumor microenvironment?

For effective multiplexed analysis of QPRT in the tumor microenvironment:

• Antibody panel design considerations:

  • Select QPRT antibodies raised in different host species from other target antibodies

  • Verify that the QPRT antibody fluorophore (e.g., CoraLite® Plus 488 in CL488-25174) has minimal spectral overlap with other fluorophores in the panel

  • Include markers for relevant cell types in the tumor microenvironment (e.g., immune cells, fibroblasts, endothelial cells)

• Sequential immunostaining protocols:

  • Implement multi-round staining with intermittent antibody stripping or quenching when direct multiplexing is challenging

  • Validate that epitope retrieval and stripping procedures do not affect QPRT detection

• Spectral unmixing and compensation:

  • Apply appropriate spectral unmixing algorithms for highly multiplexed fluorescence imaging

  • Perform single-stain controls with the QPRT antibody to establish proper compensation matrices

• Spatial analysis methodologies:

  • Combine QPRT antibody staining with digital spatial profiling technologies

  • Analyze spatial relationships between QPRT-expressing cells and other components of the tumor microenvironment

• Single-cell analysis integration:

  • Use QPRT antibodies in flow cytometry or mass cytometry (CyTOF) panels for high-dimensional single-cell analysis

  • For CL488-25174, follow the recommended concentration of 0.80 μg per 10^6 cells

• Multi-omic approaches:

  • Integrate QPRT protein detection using antibodies with transcriptomic or metabolomic analyses through sequential or parallel workflows on the same samples

How might QPRT antibodies be utilized in developing targeted therapies for QPRT-overexpressing cancers?

QPRT antibodies can contribute to therapeutic development in several ways:

• Target validation: Use QPRT antibodies to confirm differential expression between normal and cancerous tissues across large patient cohorts, establishing QPRT as a legitimate therapeutic target, particularly in HER2+ breast cancer where its expression is highest .

• Mechanism of action studies: Employ QPRT antibodies in combination with PI3K/Akt pathway inhibitors to determine whether QPRT represents a potential resistance mechanism to existing targeted therapies through activation of this pathway .

• Antibody-drug conjugate (ADC) development: If QPRT demonstrates cell-surface expression in certain contexts, explore the potential for developing QPRT-targeting therapeutic antibodies conjugated to cytotoxic payloads.

• Patient stratification biomarkers: Develop standardized immunohistochemical protocols using validated QPRT antibodies to identify patients most likely to benefit from therapies targeting QPRT-dependent pathways.

• Pharmacodynamic markers: Utilize QPRT antibodies to monitor target engagement and pathway modulation in response to experimental therapeutics targeting QPRT or related metabolic pathways.

• Combination therapy rationale: Use QPRT antibody-based assays to identify synergistic pathways for combination therapy approaches, particularly in PI3K/Akt inhibitor combinations where QPRT may influence response .

What are the methodological considerations for investigating QPRT's role in nicotinamide adenine dinucleotide (NAD+) metabolism in cancer?

To investigate QPRT's role in NAD+ metabolism in cancer:

• Activity assays coupled with expression analysis: Combine QPRT antibody-based protein quantification with enzymatic activity assays to determine whether protein expression correlates with functional activity in different cancer contexts.

• Metabolic flux analysis: Use isotope-labeled precursors to trace the contribution of QPRT-dependent pathways to NAD+ synthesis, correlating pathway activity with QPRT protein levels measured by antibody-based methods.

• Subcellular compartmentalization: Employ fractionation techniques followed by western blotting with QPRT antibodies to determine whether QPRT localizes to specific subcellular compartments where localized NAD+ synthesis may occur.

• Protein interaction network mapping: Use QPRT antibodies for immunoprecipitation followed by mass spectrometry to identify protein interaction partners involved in NAD+ metabolism regulation.

• Genetic manipulation validation: Combine CRISPR/Cas9-mediated QPRT knockout or overexpression with antibody-based detection of other NAD+ metabolism enzymes to map pathway compensations and dependencies.

• Integration with quinolinic acid metabolism: Since QPRT is involved in quinolinic acid (QA) catabolism , use QPRT antibodies alongside QA quantification assays to understand the relationship between QPRT expression and QA levels in cancer cells.

How can advanced imaging techniques enhance the utility of QPRT antibodies in understanding QPRT's dynamic behavior in living systems?

Advanced imaging approaches to study QPRT dynamics include:

• Live-cell antibody fragment imaging: Develop cell-permeable fluorescently labeled QPRT antibody fragments (e.g., Fabs) to track QPRT dynamics in living cells without genetic manipulation.

• Super-resolution microscopy: Apply techniques such as STORM, PALM, or STED using highly specific QPRT antibodies like CL488-25174 to visualize QPRT distribution at nanoscale resolution, potentially revealing previously undetected organizational patterns.

• Intravital microscopy: Utilize fluorescently conjugated QPRT antibodies for in vivo imaging of QPRT expression in tumor xenograft models to understand its dynamic regulation in the complex tumor microenvironment.

• FRET/FLIM approaches: Develop FRET pairs using QPRT antibodies and antibodies against interaction partners to visualize and quantify protein-protein interactions in situ.

• Correlative light and electron microscopy (CLEM): Combine immunofluorescence using QPRT antibodies with electron microscopy to correlate QPRT localization with ultrastructural features at nanometer resolution.

• Bioluminescence resonance energy transfer (BRET): Use QPRT antibodies in combination with luciferase-tagged potential interaction partners to monitor interactions in living systems with minimal perturbation.

• Optogenetic approaches: Integrate optogenetic control of QPRT expression or localization with antibody-based imaging to observe system responses to acute QPRT modulation.