NPC2 Antibody

Basic Research Questions

How to validate NPC2 antibody specificity in experimental models?

• Perform Western blotting with shRNA-mediated NPC2 knockdown controls (e.g., SK-Hep1 cells showing reduced 18 kDa band intensity) .

• Use peptide-blocking assays: Pre-incubate antibodies with immunogenic peptides (e.g., residues 31-40 of human NPC2) to confirm epitope specificity .

• Compare staining patterns across normal vs. cancer tissues (e.g., NPC2 downregulation in hepatoma vs. overexpression in breast cancer) .

What controls are essential for NPC2 immunohistochemical staining?

How to optimize NPC2 detection in Western blotting?

• Use 5-20% SDS-PAGE gels under reducing conditions .

• Transfer proteins to nitrocellulose membranes at 150 mA for 50-90 minutes .

• Dilute primary antibodies between 1:500–1:1000 with overnight incubation at 4°C .

Advanced Research Challenges

How to resolve discrepancies in NPC2 expression across cancer types?

• Methodological approach:

  • Compare glycosylation patterns via Western blot (e.g., altered migration in liver cancer sera) .

  • Quantify serum NPC2 levels using antigen-capture ELISA with clinical correlation (elevated in cirrhosis vs. cancer) .

  • Validate tissue-specific isoforms via 2D electrophoresis .

What strategies address cross-reactivity in non-human models?

How to investigate NPC2's role in cholesterol trafficking beyond canonical pathways?

• Apply fluorescence microscopy with DyLight®488 conjugates in live-cell imaging (e.g., HepG2 cells showing cytoplasmic localization) .

• Utilize coral NPC2 homologs (e.g., XM_021052404) to study sterol transfer mechanisms via immunoprecipitation .

• Conduct structural analyses of humanized antibody Fc regions (e.g., U.S. Pat. 5,225,539 methods) to assess cholesterol-binding domains .

Data Interpretation Guidelines

Key findings from NPC2 studies:

Antibody performance metrics: