Examining Proteasome Assembly with Recombinant Archaeal Proteasomes and Nondenaturing PAGE:  The Case for a Combined Approach

Dilrajkaur Panfair; Andrew R. Kusmierczyk

doi:10.3791/54860

JoVE Journal
Biochemistry

A subscription to JoVE is required to view this content. Sign in or start your free trial.

JoVE Journal Biochemistry

Examining Proteasome Assembly with Recombinant Archaeal Proteasomes and Nondenaturing PAGE: The Case for a Combined Approach

检查蛋白酶体大会与重组古菌蛋白酶体和非变性PAGE:该案例相结合的办法

Full Text

6,836 Views

09:57 min

December 17, 2016

DOI: 10.3791/54860-v

Dilrajkaur Panfair¹, Andrew R. Kusmierczyk¹

¹Department of Biology,Indiana University-Purdue University, Indianapolis (IUPUI)

Please note that some of the translations on this page are AI generated. Click here for the English version.

Summary

该协议使用两个亚基共表达和postlysis亚基重组蛋白酶组件的更彻底的检查混合。

Transcript

本实验的总体目标是使用重组亚基和非变性聚丙烯酰胺凝胶电泳（PAGE）分析多蛋白复合物（如蛋白酶体）的组装。这种方法可以帮助回答蛋白酶体领域的关键问题，例如，在该蛋白质复合物的组装途径中会遇到哪些中间物种。该技术的主要优点是它允许通过两种平行方法快速筛选组装，从而允许检测通路上和通路外中间体。该程序从文本协议中描述的细菌表达开始。进行细菌裂解时，将共表达所需蛋白酶体亚基组合的诱导细胞托盘在冰上解冻 5 分钟。解冻后，将托盘重悬于 600 微升裂解缓冲液中，将悬浮液在 30 度下孵育