W.M. Keck Biotechnology Resource Laboratory, Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT, USA.
Methods Mol Biol. 2021;2263:381-395. doi: 10.1007/978-1-0716-1197-5_18.
Size-exclusion chromatography (SEC) coupled with multiangle light scattering detection (SEC/MALS) enables determination of the molecular weight, oligomeric state, and stoichiometry of protein-nucleic acid complexes in solution. Often such complexes show anomalous behavior on SEC, thus presenting a challenge in determination of molecular weight and stoichiometry based solely on the elution position from SEC. In contrast to analytical ultracentrifugation, the SEC/MALS analysis is not affected by the shape of the complex. Here we describe the use of SEC/MALS for characterization of the stoichiometry of the complex between the reverse transcriptase (RT) domain from group II intron-maturase from Eubacterium rectale and intron RNA, and for monitoring protein dimerization that is driven by interaction between single-stranded DNA upstream of the P1 promoter, known as FUSE and FUSE binding protein-interacting repressor (FIR).
体积排阻色谱（SEC）与多角度光散射检测（SEC/MALS）相结合，可用于确定溶液中蛋白质-核酸复合物的分子量、寡聚状态和化学计量比。通常，此类复合物在 SEC 上表现出异常行为，因此仅基于 SEC 的洗脱位置来确定分子量和化学计量比具有一定挑战性。与分析超速离心法不同，SEC/MALS 分析不受复合物形状的影响。本文描述了 SEC/MALS 在鉴定来自直肠真杆菌的内含子-成熟酶的 RT 结构域与内含子 RNA 之间复合物的化学计量比中的应用，以及用于监测由 P1 启动子上游的单链 DNA （称为 FUSE）与 FUSE 结合蛋白相互作用的阻遏物（FIR）之间的相互作用驱动的蛋白质二聚化的应用。
