Common Pitfalls and Recommendations for Using a Turbidity Assay to Study Protein Phase Separation

Area of Science:

• Biochemistry
• Biophysics
• Molecular Biology

Background:

• Turbidity assays are widely used to study protein liquid-to-liquid phase separation (LLPS) and liquid-to-solid phase separation (LSPS).
• These assays can yield inaccurate quantification when the detection linear range is exceeded.
• Aggregated proteins with high concentration and large particle size are primary causes of assay limitations.

Purpose of the Study:

• To identify common pitfalls in turbidity assays related to exceeding the linear detection range.
• To demonstrate how protein aggregation affects quantification in various biochemical applications.
• To propose simple methods for improving the accuracy of turbidity assays for studying protein phase separation.

Main Methods:

• Investigated the impact of high protein concentration and large particle size on turbidity measurements.
• Evaluated the influence of these factors on optical density measurements, thermal shift assays, and dynamic light scattering experiments.
• Assessed the effectiveness of sample dilution and direct imaging for correcting assay inaccuracies.

Main Results:

• High concentrations and large particle sizes of aggregated proteins lead to inaccurate quantification in turbidity-based assays.
• The limitations were observed across multiple applications, including optical density, thermal shift, and dynamic light scattering.
• Sample dilution for LSPS and direct imaging for LLPS were effective in addressing these inaccuracies.

Conclusions:

• Exceeding the linear detection range is a significant pitfall in turbidity assays for protein phase separation.
• Protein aggregation, characterized by high concentration and large particle size, compromises assay accuracy.
• Simple adjustments like sample dilution or direct imaging can enhance the reliability of turbidity assays for studying LLPS and LSPS.