NPLOC4 Antibody

What are the optimal applications for NPLOC4 antibody in cellular protein detection?

NPLOC4 antibody has been successfully validated across multiple applications with specific optimization parameters. Western blot (WB) remains the most widely used application with established dilution ranges of 1:500-1:2000. Immunohistochemistry (IHC) typically requires dilutions of 1:20-1:200, while immunofluorescence (IF) and immunocytochemistry (ICC) perform optimally at 1:50-1:200 dilutions . Immunoprecipitation (IP) applications require 0.5-4.0 μg of antibody per 1.0-3.0 mg of total protein lysate . For all applications, it is strongly recommended to titrate the antibody concentration for each specific experimental system and cell/tissue type to achieve optimal signal-to-noise ratios.

What tissue and species reactivity should researchers expect when using NPLOC4 antibodies?

Most commercially available NPLOC4 antibodies show validated reactivity across human, mouse, and rat samples . Positive Western blot detection has been specifically documented in mouse kidney tissue, mouse heart tissue, and rat heart tissue . For immunohistochemistry, human kidney tissue shows strong positive signal . Researchers working with non-mammalian systems should note that some antibodies are specifically designed for detection in yeast (Saccharomyces cerevisiae, Schizosaccharomyces) and Drosophila melanogaster systems . Always verify species cross-reactivity in the product datasheet before designing experiments with novel tissue or species combinations.

What is the recommended storage protocol for maintaining NPLOC4 antibody activity?

For long-term storage, NPLOC4 antibodies should be maintained at -20°C, where they typically remain stable for one year after shipment . Most preparations contain glycerol (commonly 50%) with 0.02% sodium azide in PBS at pH 7.3 to prevent freeze-thaw damage . For frequent use over short periods (up to one month), storage at 4°C is acceptable . Aliquoting is generally unnecessary for -20°C storage for standard preparations, though some manufacturers recommend it for preparations without sufficient cryoprotectant . When working with small volume preparations (20μl), note that some formulations contain 0.1% BSA as an additional stabilizer .

How should researchers optimize NPLOC4 antibody protocols for detecting subcellular localization changes?

NPLOC4 exhibits complex subcellular localization patterns across the nucleus, endoplasmic reticulum, and cytoplasm , requiring careful optimization for accurate detection. For immunofluorescence studies, use fixation with 4% paraformaldehyde followed by permeabilization with 0.2% Triton X-100. Antibody concentrations should be optimized between 0.25-2 μg/mL . When studying NPLOC4's dynamic localization during cell cycle progression, particularly during mitosis when it participates in spindle disassembly, time-course experiments with synchronized cell populations are recommended .

For colocalization studies examining NPLOC4's interaction with VCP and UFD1L in the ubiquitin-proteasome pathway, use confocal microscopy with appropriate spectral separation between fluorophores. When assessing nuclear envelope localization, super-resolution techniques such as STED or STORM microscopy provide better resolution of the nuclear membrane association pattern than standard confocal approaches.

What controls and validation steps are necessary when using NPLOC4 antibody for studying its role in cancer progression?

When investigating NPLOC4 as a prognostic marker in cancer research (particularly lung squamous cell carcinoma), multiple validation steps are essential:

• Positive tissue controls: Include known positive samples such as human kidney tissue for IHC or mouse kidney for WB and IP .

• Expression knockout/knockdown controls: NPLOC4 knockdown/knockout systems provide crucial negative controls, with several published protocols available .

• Scoring system standardization: For IHC quantification, implement a standardized scoring system that accounts for both staining intensity (0-4 scale) and the fraction of positive cells, as described in published protocols .

• Antibody validation criteria:

  Validation Parameter	Recommended Approach	Key Considerations
  Specificity	Western blot showing expected 68 kDa band	Verify absence of non-specific bands
  Sensitivity	Titration experiments	Determine minimal detectable expression level
  Reproducibility	Technical replicates	Consistent results across experiments
  Cross-reactivity	Testing across species	Confirm reactivity matches product claims
  Epitope mapping	Immunogen sequence analysis	Understanding binding region may explain results

• Correlation with patient data: When examining prognostic value, NPLOC4 expression should be evaluated in conjunction with TNM staging and lymph node metastasis status, as higher NPLOC4 expression correlates with stages III-IV and lymph node metastasis .

What methodological approaches should be used when investigating NPLOC4's role in the ubiquitin-proteasome pathway?

To study NPLOC4's function in ubiquitin-mediated proteolysis:

• K48-linked ubiquitin detection: Use anti-K48-ubiquitin antibodies (1:10,000 dilution) in conjunction with NPLOC4 antibodies to assess accumulation of polyubiquitinated proteins following NPLOC4 inhibition .

• DSF+Cu treatment model: When studying disulfiram (DSF) and copper (Cu) effects on NPLOC4, optimize concentrations (0.25 μM DSF and 0.2 μM Cu for 24h has shown efficacy in SK-MES-1 cell lines) and verify by measuring apoptosis markers and K48-linked ubiquitinated protein levels .

• Protein complex analysis: For studying the NPLOC4-UFD1L-VCP complex, use co-immunoprecipitation with NPLOC4 antibody (0.5-4.0 μg per IP reaction) followed by western blot detection of complex components .

• Gene-set enrichment analysis: When examining pathway involvement, use GSEA with KEGG datasets as demonstrated in published protocols, with at least 1000 iterations and significance filtering (p < 0.05, FDR < 0.05) .

What are the key molecular characteristics of NPLOC4 protein that researchers should consider when selecting antibodies?

NPLOC4 protein has several structural and molecular features that influence antibody selection:

When selecting antibodies, researchers should consider whether their experimental questions require detection of specific domains or post-translational modifications of NPLOC4. The C-terminal region (amino acids 456-484) is commonly used as an immunogen for antibody production and provides good specificity .

How should researchers evaluate NPLOC4 antibody performance across different experimental systems?

Systematic antibody evaluation should include:

• Sensitivity assessment: Determine the lower limit of detection through serial dilutions of positive control lysates (mouse kidney tissue is recommended) .

• Cross-application performance: If using the same antibody across multiple applications (WB, IHC, IP), validate each application independently using recommended dilutions:

  • Western Blot: 1:500-1:1000

  • Immunohistochemistry: 1:20-1:200

  • Immunoprecipitation: 0.5-4.0 μg per reaction

• ROC curve analysis: For diagnostic applications, generate ROC curves to evaluate the antibody's discriminatory power between disease and normal states. Published studies show NPLOC4 expression can discriminate between LUSC and normal lung tissues with AUC values of 0.930-0.995 .

• Technical replicates: Minimum of three technical replicates are recommended for quantitative analyses, particularly for immunohistochemistry scoring where multiple fields should be evaluated and averaged .

• Antibody validation reporting: Document all validation parameters according to the International Working Group for Antibody Validation (IWGAV) guidelines.

How can NPLOC4 antibodies be effectively utilized to evaluate cancer prognosis and therapeutic response?

NPLOC4 serves as both a prognostic biomarker and potential therapeutic target, particularly in lung squamous cell carcinoma:

• Prognostic evaluation protocol:

  • Stratify patient samples based on NPLOC4 expression (high/low) using IHC with 1:20-1:200 antibody dilution

  • Use Kaplan-Meier survival analysis to correlate expression with patient outcomes

  • Validate prognostic value using ROC curves for 1, 3, 5, and 10-year survival predictions

  • Perform multivariate Cox regression analysis including NPLOC4 expression, age, TNM stage, and AJCC stage

• Therapeutic response monitoring:

  • When evaluating DSF+Cu treatment efficacy, measure NPLOC4 protein levels along with K48-linked ubiquitin accumulation

  • Assess correlation between NPLOC4 expression reduction and apoptosis markers

  • Monitor changes in NPLOC4 localization during treatment using immunofluorescence

What methodological approaches are recommended for studying NPLOC4's interaction with the tumor immune microenvironment?

The relationship between NPLOC4 expression and tumor-infiltrating immune cells (TICs) involves several methodological considerations:

• Correlation analysis protocol:

  • Use CIBERSORT algorithm to estimate TIC abundance in tumor samples (select samples with p < 0.05)

  • Analyze correlation between NPLOC4 expression and specific immune cell populations

  • Utilize the TIMER database (https://cistrome.shinyapps.io/timer/) to evaluate correlation between NPLOC4 and immune checkpoints

• Multiplex immunofluorescence approach:

  • Use NPLOC4 antibody (1:50-1:200) in combination with immune cell markers

  • Implement spectral unmixing to resolve overlapping fluorophore signals

  • Analyze spatial relationships between NPLOC4-expressing tumor cells and immune infiltrates

• Immune checkpoint correlation:

  • Assess co-expression patterns of NPLOC4 with PD-L1, PD-1, CTLA-4, and other checkpoint molecules

  • Evaluate potential mechanistic links through pathway analysis

This approach provides insight into how NPLOC4 expression may influence immunotherapy response, potentially identifying patient subgroups who might benefit from combined NPLOC4-targeting and immunotherapy approaches.

How can researchers design experiments to investigate NPLOC4's role beyond cancer in other pathological conditions?

While NPLOC4's role in cancer (particularly LUSC) is established, its function in other conditions remains underexplored. To investigate broader roles:

• Comparative tissue analysis protocol:

  • Use tissue microarrays with NPLOC4 antibody (1:20-1:200) across multiple disease states

  • Implement standardized scoring system (0-4 intensity × percentage positive cells)

  • Compare expression patterns across neurodegenerative disorders, inflammatory conditions, and metabolic diseases

• Cellular stress response studies:

  • Given NPLOC4's role in the ubiquitin-proteasome pathway, examine expression changes during ER stress

  • Monitor NPLOC4 localization during oxidative stress using immunofluorescence (1:50-1:200)

  • Investigate potential roles in proteotoxic stress conditions like those found in neurodegenerative disorders

• NPLOC4 complex formation assessment:

  • Analyze the NPLOC4-UFD1L-VCP complex in different pathological states using co-immunoprecipitation

  • Compare complex formation and stability across normal and disease conditions

  • Evaluate potential therapeutic approaches targeting this complex in diverse disease contexts

These approaches expand NPLOC4 research beyond its established cancer role, potentially identifying novel therapeutic applications in other diseases.

What technical considerations are essential when developing high-throughput screening assays using NPLOC4 antibodies?

For researchers developing screening assays to identify NPLOC4 modulators or for large-scale patient sample analysis:

• Assay development considerations:

  • Antibody selection: Choose antibodies with highest specificity and lowest batch variability

  • Assay format: ELISA-based assays using NPLOC4 antibody (optimize coating concentration between 1-10 μg/mL)

  • Signal detection: HRP-conjugated secondary antibodies provide better sensitivity than fluorescence in plate-based formats

• Validation requirements:

  • Z'-factor determination: Ensure Z' > 0.5 for reliable screening performance

  • LLOD/LLOQ establishment: Determine lower limits of detection and quantification

  • Standard curve generation: Use recombinant NPLOC4 protein for calibration

• Cell-based screening approach:

  • Reporter system: Consider developing NPLOC4-GFP fusion constructs for live-cell imaging

  • High-content analysis: Use automated image analysis to quantify NPLOC4 localization changes

  • Phenotypic endpoints: Correlate NPLOC4 modulation with functional outcomes (ubiquitinated protein accumulation, cell viability)

These technical considerations ensure development of robust, reproducible high-throughput assays that can accelerate NPLOC4-focused drug discovery and biomarker validation.