Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

Nicholas McCaul, Hui Ying Yeoh, Guus van Zadelhoff, Naomi Lodder, Bertrand Kleizen, Ineke Braakman

Research output: Contribution to journalArticlepeer-review

5 Citations (Scopus)

Abstract

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and the degradation of target proteins in live cells. By using short (pulse) radiolabeling times (<30 min) and tightly controlled chase times, it is possible to label only a small fraction of the total protein pool and follow its folding. When combined with nonreducing/reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation with (conformation-specific) antibodies, folding processes can be examined in great detail. This system has been used to analyze the folding of proteins with a huge variation in properties such as soluble proteins, single and multi-pass transmembrane proteins, heavily N- and O-glycosylated proteins, and proteins with and without extensive disulfide bonding. Pulse-chase methods are the basis of kinetic studies into a range of additional features, including co- and posttranslational modifications, oligomerization, and polymerization, essentially allowing the analysis of a protein from birth to death. Pulse-chase studies on protein folding are complementary with other biochemical and biophysical methods for studying proteins in vitro by providing increased temporal resolution and physiological information. The methods as described within this paper are adapted easily to study the folding of almost any protein that can be expressed in mammalian or insect-cell systems.

Original languageEnglish
Article numbere58952
JournalJournal of visualized experiments : JoVE
Issue number144
DOIs
Publication statusPublished - 12 Feb 2019
Externally publishedYes

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