pfk-1.1 Antibody

FAQs for PFK-1.1 Antibody in Academic Research

How is PFK-1.1 antibody specificity validated in functional assays?

Methodological validation involves:

• Knockdown confirmation: Use RNAi constructs (e.g., shRNA) targeting PFK-1.1, followed by RT-PCR and Western blot to quantify mRNA/protein reduction. In CNE2 nasopharyngeal carcinoma cells, sh-PFK-507 reduced PFK1 mRNA by 4.0-fold (p < 0.001) and enzyme activity by 3.0-fold .

• Immunofluorescence controls: Include isotype-matched antibodies and untransfected cells to assess non-specific binding .

• Functional rescue experiments: Re-express PFK-1.1 in knockdown models to reverse phenotypic effects (e.g., restored glycolysis in PFK1-deficient cells) .

What experimental controls are critical for PFK-1.1 antibody-based assays?

• Negative controls: Untransfected cells, scrambled shRNA, and isotype-matched antibodies .

• Positive controls: Tissues/cells with confirmed high PFK-1.1 expression (e.g., metastatic cancer lines) .

• Technical replicates: Run assays in triplicate to account for plate variability in ELISA or Western blot .

How to design RNAi experiments using PFK-1.1 antibody in cancer models?

• Construct selection: Test multiple shRNA sequences (e.g., sh-PFK-507 achieved 75% knockdown efficiency in CNE2 cells vs. non-functional sh-PFK-698) .

• Phenotypic assays:

  • Migration/Invasion: Use wound healing and Boyden chamber assays. PFK-1.1 knockdown reduced CNE2 cell invasion by 60% .

  • Apoptosis: Measure caspase-3/7 activity; PFK-1.1 silencing increased apoptosis by 2.5-fold .

Table 1: shRNA Knockdown Efficiency in CNE2 Cells

How to resolve discrepancies in PFK-1.1 expression data across studies?

• Source validation: Confirm antibody cross-reactivity using KO cell lines .

• Contextual factors:

  • Tissue specificity: PFK-1.1 is upregulated in metastatic nasopharyngeal carcinoma but may vary in other cancers .

  • Assay thresholds: Optimize ELISA cutoffs using receiver operating characteristic (ROC) curves .

Can PFK-1.1 inhibition synergize with PD-1/PD-L1 blockade in immunotherapy?

• Mechanistic rationale: PFK-1.1 regulates glycolysis, which impacts T-cell metabolism and tumor microenvironment .

• Experimental design:

  • Combination therapy: Administer PFK-1.1 siRNA with anti-PD-L1 antibodies (e.g., atezolizumab) in murine models .

  • Outcome metrics: Measure tumor volume, CD8+ T-cell infiltration, and IFN-γ levels .

Methodological Best Practices

• Antibody validation: Follow OIE Validation Standards for specificity, repeatability, and robustness .

• Data normalization: Use housekeeping genes (e.g., GAPDH) for Western blot and spike-in controls for ELISA .

Table 2: Key Parameters for PFK-1.1 Antibody Validation