Abstract
This chapter describes the assays for Rab3 guanine nucleotide exchange protein (GEP) activity, the procedures for the purification of native Rab3 GEP from rat brain, the procedures for the purification of recombinant Rab3 GEP from COS7 cells, and the properties of Rab3 GEP. The Rab3 GEP activity to stimulate the GDP/GTP exchange reaction of Rab3A is assayed by measuring either the dissociation of [ 3 H]GDP from or binding of [ 35 S]GTPTS to lipid-modified Rab3A. The steps used in the purification of Rab3 GEP from rat brain are: (1) preparation of the synaptic soluble fraction from rat brain, (2) Q-Sepharose FF column chromatography, (3) phenyl-Sepharose column chromatography, (4) hydroxyapatite column chromatography, (5) Mono Q HR10/10 column chromatography, (6) HiLoad 16/60 Superdex 200 column chromatography, and (7) HPLC hydroxyapatite column chromatography. All the purification procedures are carried out at 0–4°C.
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