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

Biochemistry. 2021-08; 
Yanan Huang, Yulong Bai, Wenhan Jin, Di Shen, Haochen Lyu, Lianggang Zeng, Mengdie Wang, Yu Liu
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Codon Optimization … coli wild-type dihydrofolate reductase (WT-DHFR) and sortase were codon optimized, synthesized by GenScript, and subcloned into pET-29b (+) … coli transthyretin L55P mutant were codon optimized, synthesized by GenScript, and subcloned into pET-29b (+) vectors. TTR-L55P … Get A Quote

摘要

The turbidity assay is commonly exploited to study protein liquid-to-liquid phase separation (LLPS) or liquid-to-solid phase separation (LSPS) processes in biochemical analyses. Herein, we present common pitfalls of this assay caused by exceeding the detection linear range. We showed that aggregated proteins of high concentration and large particle size can lead to inaccurate quantification in multiple applications, including the optical density measurement, the thermal shift assay, and the dynamic light scattering experiment. Finally, we demonstrated that a simple sample dilution of insoluble aggregated protein (LSPS) samples or direct imaging of liquid droplets (LLPS) can address these issues and improve the ... More

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