Pulse-Chase Analysis for Studies of MHC Class II Biosynthesis, Maturation, and Peptide Loading.
Pulse-Chase Analysis for Studies of MHC Class II Biosynthesis, Maturation, and Peptide Loading.
复制标题
用于 MHC II 类生物合成、成熟和肽加载研究的脉冲追踪分析。
DOI：
10.1007/978-1-4939-9450-2_23
复制
发表时间：
2019
期刊：
影响因子：
--
通讯作者：
Mellins,ElizabethD
中科院分区：
文献类型：
--
作者：
Hou,Tieying;Rinderknecht,Cornelia;Ghosh,Debopam;Hadjinicolaou,AndreasV;Busch,Robert;Mellins,ElizabethD
脉冲追踪分析是研究蛋白质合成、加工和转运的常用技术。使表达感兴趣的蛋白质的培养细胞摄取放射性标记的氨基酸一段短暂的时间（“脉冲”），在此期间所有新合成的蛋白质都掺入了标记。然后将细胞返回到非放射性培养基中不同的时间（“追踪”），在此期间蛋白质可能经历构象变化、运输或降解。分离（通常通过免疫沉淀法）目标蛋白，并通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）进行分离，并通过放射自显影法检查放射性标记分子的命运。本章介绍了一个脉冲追逐协议适用于主要组织相容性复合体（MHC）II类生物合成和成熟的研究。我们讨论了结果是如何受到某些抗II类抗体的识别与特定的生物合成状态相关的不同的II类构象。我们的协议可以适应于遵循许多其他内源性合成的蛋白质，包括病毒或转染的基因产物，在培养细胞的命运。
Pulse–chase analysis is a commonly used technique for studying the synthesis, processing, and transport of proteins. Cultured cells expressing proteins of interest are allowed to take up radioactively labeled amino acids for a brief interval (“pulse”), during which all newly synthesized proteins incorporate the label. The cells are then returned to nonradioactive culture medium for various times (“chase”), during which proteins may undergo conformational changes, trafficking, or degradation. Proteins of interest are isolated (usually by immunoprecipitation) and resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and the fate of radiolabeled molecules is examined by autoradiography. This chapter describes a pulse–chase protocol suitable for studies of major histocompatibility complex (MHC) class II biosynthesis and maturation. We discuss how results are affected by the recognition by certain anti-class II antibodies of distinct class II conformations associated with particular biosynthetic states. Our protocol can be adapted to follow the fate of many other endogenously synthesized proteins, including viral or transfected gene products, in cultured cells.