title: Pulse-chase analysis for studying protein synthesis and maturation.
abstract: Pulse-chase analysis is a well-established and highly adaptable tool for studying the life cycle of endogenous proteins, including their synthesis, folding, subunit assembly, intracellular transport, post-translational processing, and degradation. This unit describes the performance and analysis of a radiolabel pulse-chase experiment for following the folding and cell surface trafficking of a trimeric murine MHC class I glycoprotein. In particular, the unit focuses on the precise timing of pulse-chase experiments to evaluate early/short-time events in protein maturation in both suspended and strictly adherent cell lines. The advantages and limitations of radiolabel pulse-chase experiments are discussed, and a comprehensive section for troubleshooting is provided. Further, ways to quantitatively represent pulse-chase results are described, and feasible interpretations on protein maturation are suggested. The protocols can be adapted to investigate a variety of proteins that may mature in very different ways.
authors.affiliation: Department of Life Sciences and Chemistry, Jacobs University Bremen, Bremen, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Fritzsche
authors.affiliation: Department of Life Sciences and Chemistry, Jacobs University Bremen, Bremen, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Springer
mesh_terms: D000818:Animals; D015395:Histocompatibility Antigens Class I / Q000096:biosynthesis*; D007553:Isotope Labeling / Q000379:methods*; D051379:Mice; D014176:Protein Biosynthesis / Q000502:physiology*; D017510:Protein Folding
chemical_list: D015395:Histocompatibility Antigens Class I
keywords: glycan processing; immunoprecipitation; metabolic labeling; protein folding; protein transport; secretory pathway