Hou Tieying, Rinderknecht Cornelia H, Hadjinicolaou Andreas V, Busch Robert, Mellins Elizabeth
Department of Pediatrics, Division of Transplantation Biology and Immunology, Stanford University Medical School, Stanford, CA, USA.
Division of Transplantation Biology and Immunology, Department of Pediatrics, Stanford University Medical School, Stanford, CA, USA.
Methods Mol Biol. 2013;960:411-432. doi: 10.1007/978-1-62703-218-6_31.
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.
脉冲追踪分析是一种常用的技术，用于研究蛋白质的合成、加工和运输。使表达感兴趣蛋白质的培养细胞在短时间间隔内摄取放射性标记的氨基酸（“脉冲”），在此期间所有新合成的蛋白质都掺入该标记。然后将细胞放回无放射性的培养基中培养不同时间（“追踪”），在此期间蛋白质可能会发生构象变化、运输或降解。分离出感兴趣的蛋白质（通常通过免疫沉淀法），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，通过放射自显影检查放射性标记分子的命运。本章描述了一种适用于研究主要组织相容性复合体（MHC）II类生物合成和成熟的脉冲追踪方案。我们讨论了某些抗II类抗体对与特定生物合成状态相关的不同II类构象的识别如何影响结果。我们的方案可适用于追踪培养细胞中许多其他内源性合成蛋白质的命运，包括病毒或转染基因产物。
