Protocol for single-molecule pull-down of fluorescently tagged oligomers from cell lysates.
Protocol for single-molecule pull-down of fluorescently tagged oligomers from cell lysates.
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DOI：
10.1016/j.xpro.2023.102716
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发表时间：
2023-12-15
期刊：
影响因子：
--
通讯作者：
Myong, Sua
中科院分区：
文献类型：
--
作者：
Djaja, Nathalie;Myong, Sua
本质上无序的蛋白质的突变会导致病理聚集体的不可逆形成，这是神经退行性疾病的标志。在这里，我们提出了一种拉下荧光标记蛋白质以表征其基础寡聚状态的方案。我们描述了转染和细胞裂解、单分子载玻片制备和下拉以及寡聚物溶解的步骤。该协议能够以单分子分辨率可视化蛋白质寡聚体。此外，寡聚化的差异可以提供对不同突变或细胞应激条件下的缩合或聚集倾向的了解。有关该协议的使用和执行的完整详细信息，请参阅 Djaja 等人。从细胞裂解液中分离和下拉基础蛋白寡聚物 溶解蛋白寡聚物，以了解寡聚物的形成 用于单分子下拉的载玻片制备和组装的详细步骤 基础蛋白寡聚化，以提供有关蛋白质缩合的见解 出版商注：进行任何实验方案都需要遵守当地实验室安全和道德机构指南。本质上无序的蛋白质的突变会导致病理聚集体的不可逆形成，这是神经退行性疾病的标志。在这里，我们提出了一种拉下荧光标记蛋白质以表征其基础寡聚状态的方案。我们描述了转染和细胞裂解、单分子载玻片制备和下拉以及寡聚物溶解的步骤。该协议能够以单分子分辨率可视化蛋白质寡聚体。此外，寡聚化的差异可以提供对不同突变或细胞应激条件下的缩合或聚集倾向的了解。
Mutations in intrinsically disordered proteins drive the irreversible formation of pathological aggregates, a hallmark of neurodegenerative diseases. Here, we present a protocol to pull down fluorescently tagged proteins to characterize their basal oligomeric states. We describe steps for transfection and cell lysis, single-molecule slide preparation and pull-down, and oligomer dissolution. This protocol enables visualization of protein oligomers with single-molecule resolution. In addition, differences in oligomerization may provide insight on condensation or aggregation propensity in differing mutated or cell stress conditions. For complete details on the use and execution of this protocol, please refer to Djaja et al. Isolation and pull-down of basal protein oligomers from cell lysates Dissolution of protein oligomers to provide insight on oligomer formation Detailed steps for slide preparation and assembly for single-molecule pull-down Basal protein oligomerization to provide insight on protein condensation Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Mutations in intrinsically disordered proteins drive the irreversible formation of pathological aggregates, a hallmark of neurodegenerative diseases. Here, we present a protocol to pull down fluorescently tagged proteins to characterize their basal oligomeric states. We describe steps for transfection and cell lysis, single-molecule slide preparation and pull-down, and oligomer dissolution. This protocol enables visualization of protein oligomers with single-molecule resolution. In addition, differences in oligomerization may provide insight on condensation or aggregation propensity in differing mutated or cell stress conditions.
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DOI：
10.3791/50549
发表时间：
2014-04-24
期刊：
Journal of visualized experiments : JoVE
影响因子：
--
作者：
Chandradoss SD;Haagsma AC;Lee YK;Hwang JH;Nam JM;Joo C
通讯作者：
Joo C
影响因子：
5.8
作者：
Djaja, Nathalie A.;Chang, Matthew T.;Morris, Freya R.;Morris, Vivian M.;Ganser, Laura R.;Myong, Sua
通讯作者：
Myong, Sua
影响因子：
--
作者：
Joo, Chirlmin;Ha, Taekjip
通讯作者：
Ha, Taekjip
影响因子：
--
作者：
Paul T;Myong S
通讯作者：
Myong S