Abstract
This chapter discusses and presents the assay system and the procedures needed to isolate and purify the active domain of the docking protein from isolated rough microsomes. The processing of nascent secretory proteins involves cotranslational cleavage of the signal sequence on the luminal side of microsomal vesicles. The general principle of using dog pancreas microsomes to reconstitute translocation of secretory proteins must be slightly modified to allow the addition of isolated factors to the system and the membranes. Purification of docking protein involves preparation of stripped rough microsomes, detergent extraction of stripped rough microsomes (RM), elastase extraction of triton-insoluble membrane material, purification of the fragment of docking protein (DP f ) from elastase extract (EE) using ion-exchange chromatography, and gel filtration of CM-bound EE. The protein composition of fractions is best determined by polyacrylamide gel electrophoresis and staining with either Coomassie Blue or silver.
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