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Semipermeabilized Cells to Study Procollagen Assembly

  • Richard R. Wilson
  • Neil J. Bulleid
Part of the Methods in Molecular Biology™ book series (MIMB, volume 139)

Abstract

This chapter will describe the preparation and use of a semipermeabilized (SP) cell system that reconstitutes the initial stages in the assembly and modification of proteins entering the secretory pathway (1). The procedure involves treating cells grown in culture with the detergent digitonin and isolating the cells free from their cytosolic component (2). The expression of proteins in an SP cell system allows protein assembly to be studied in an environment that more closely resembles that of the intact cell. As this is an in vitro system, the individual components can be manipulated easily, providing a means by which cellular processes can be studied under a variety of conditions. In addition, membrane-permeable chemical crosslinking reagents can be added in order to facilitate the study of interaction between proteins within the endoplasmic reticulum (ER) lumen (1,3). Furthermore, as the ER remains morphologically intact, the spatial localization of folding and transport processes within the reticular network may also be examined.

Keywords

Endoplasmic Reticulum HT1080 Cell Endoplasmic Reticulum Membrane Endoplasmic Reticulum Lumen Rabbit Reticulocyte Lysate 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Humana Press Inc. 2000

Authors and Affiliations

  • Richard R. Wilson
    • 1
  • Neil J. Bulleid
    • 1
  1. 1.School of Biological SciencesUniversity of ManchesterManchesterUK

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