分泌物
跨膜蛋白
聚丙烯酰胺凝胶电泳
膜
化学
膜蛋白
功能(生物学)
质谱法
生物化学
基质(水族馆)
三型分泌系统
凝胶电泳
生物
细胞生物学
色谱法
毒力
酶
生态学
受体
基因
作者
Susann Zilkenat,Eun‐jin Kim,Tobias Dietsche,Julia Monjarás Feria,Claudia E. Torres‐Vargas,Mehari Tesfazgi Mebrhatu,Samuel Wagner
标识
DOI:10.1007/978-1-0716-3445-5_22
摘要
Bacterial protein secretion systems serve to translocate substrate proteins across up to three biological membranes, a task accomplished by hydrophobic, membrane-spanning macromolecular complexes. The overexpression, purification, and biochemical characterization of these complexes is often difficult, thus impeding progress in understading structure and function of these systems. Blue native (BN) polyacrylamide gel electrophoresis (PAGE) allows for the investigation of these transmembrane complexes right from their originating membranes, without the need of long preparative steps, and is amenable to the parallel characterization of a number of samples under near-native conditions. Here, we present protocols for sample preparation, one-dimensional BN PAGE and two-dimensional BN/SDS PAGE, as well as for downstream analysis by staining, immunoblotting, and mass spectrometry on the example of the type III secretion system encoded on Salmonella pathogenicity island 1.
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