Analysis of protein folding and oxidation in the endoplasmic reticulum.

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.