Abstract
This chapter discusses the purification of TRAPP from Saccharomyces cerevisiae and identification of its mammalian counterpart. Primarily construction of Bet3p-protein A fusion used to purify TRAPP is discussed. TRAPP is initially purified using a strain in which the sole copy of Bet3p is tagged at its carboxy terminus with three c-myc epitopes. The Bet3p-associated proteins are then purified from this strain by precipitating the complex from a lysate. This is done by incubating a clarified detergent extract with affinity purified anti- c-myc antibody bound to Affi-Gel. The bound protein is eluted and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This affinity purification led to the identity of several TRAPP subunits. In addition, large-scale purification of TRAPP and identification of TRAPP subunits, small-scale precipitation of TRAPP, and identification of mammalian TRAPP from HeLa lysates are described.
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