Unit

UNIT 15.6 Analysis of Protein Folding and Oxidation in the Endoplasmic Reticulum

  1. Edwin Francis,
  2. Robert Daniels,
  3. Daniel N. Hebert

Published Online: 1 MAY 2002

DOI: 10.1002/0471143030.cb1506s14

Current Protocols in Cell Biology

Current Protocols in Cell Biology

How to Cite

Francis, E., Daniels, R. and Hebert, D. N. 2002. Analysis of Protein Folding and Oxidation in the Endoplasmic Reticulum. Current Protocols in Cell Biology. 14:15.6:15.6.1–15.6.29.

Author Information

  1. University of Massachusetts, Amherst, Massachusetts

Publication History

  1. Published Online: 1 MAY 2002
  2. Published Print: APR 2002

Abstract

Proteins that travel through the secretory pathway undergo post-translational folding and oxidation steps that lead to correct conformation of the final protein. This unit focuses on methods for the analysis of folding and oxidation events and the factors responsible for their proper execution. Alkylation and nonreducing SDS-PAGE is use to analyze disulfide bond formation in ER-derived microsomes. If proteins are synthesized under reducing conditions, it is possible to initiate folding by addition of oxidizing agents, thus allowing analysis of factors necessary for the folding process. As folding progresses, the protein of interest shows a change in sensitivity to proteolysis. Co-immunoprecipitation or crosslinking and denaturing immunoprecipitation are used to explore the role of molecular chaperones and other factors. Conformation-specific antibodies can be used to probe folding. In addition, folding can be analyzed in intact or semi-permeabilized adherent or suspension cells.