Suppressing Nonspecific Binding in Biolayer Interferometry Experiments for Weak Ligand-Analyte Interactions

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Quantitative analysis of protein-protein interactions (PPIs) via biolayer interferometry (BLI) necessitates minimizing nonspecific binding (NSB).
• High analyte concentrations, often required for studying weak interactions (K_D > 1 μM), exacerbate NSB.
• Limited availability of biomolecule-compatible NSB blockers restricts accurate BLI analysis of weak interactions.

Purpose of the Study:

• To identify a novel NSB blocker for quantitative analysis of weak PPIs using BLI.
• To evaluate the efficacy and compatibility of saccharides, particularly sucrose, as NSB blockers.

Main Methods:

• Biolayer interferometry (BLI) was employed for quantitative analysis of PPIs.
• Sucrose was investigated as a potential NSB blocker.
• The binding kinetics between influenza A virus nonstructural protein 1 and human phosphoinositide 3-kinase were characterized.

Main Results:

• Sucrose demonstrated potent NSB blocking capabilities in BLI.
• Sucrose proved compatible with other blocking additives.
• The study successfully characterized weak interactions using sucrose as an NSB blocker.

Conclusions:

• Sucrose is an effective NSB blocker for BLI, particularly for weak interactions.
• The use of sucrose enhances the accuracy of quantitative PPI analysis using BLI.
• This novel NSB-blocking strategy is expected to have wide applications in studying weak molecular interactions.