重组DNA
化学
配体(生物化学)
变性(裂变材料)
靶蛋白
荧光
蛋白质稳定性
高通量筛选
生物物理学
色谱法
热稳定性
生物化学
生物
有机化学
受体
量子力学
基因
物理
核化学
作者
Kathy Huynh,Carrie L. Partch
标识
DOI:10.1002/0471140864.ps2809s79
摘要
Abstract Purification of recombinant proteins for biochemical assays and structural studies is time‐consuming and presents inherent difficulties that depend on the optimization of protein stability. The use of dyes to monitor thermal denaturation of proteins with sensitive fluorescence detection enables rapid and inexpensive determination of protein stability using real‐time PCR instruments. By screening a wide range of solution conditions and additives in a 96‐well format, the thermal shift assay easily identifies conditions that significantly enhance the stability of recombinant proteins. The same approach can be used as an initial low‐cost screen to discover new protein‐ligand interactions by capitalizing on increases in protein stability that typically occur upon ligand binding. This unit presents a methodological workflow for small‐scale, high‐throughput thermal denaturation of recombinant proteins in the presence of SYPRO Orange dye. © 2015 by John Wiley & Sons, Inc.
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