Abstract
This chapter describes the assays for the Rab3 guanosine-5'-triphosphate (GTP)ase-activating proteins (GAP) activity, the procedures for the purification of native Rab3 GAP from rat brain synaptic soluble fraction, the procedures for the purification of recombinant hexahistidine (His6)-tagged Rab3 GAP from Escherichia coli, and the properties of Rab3 GAP. The Rab3 GAP activity to stimulate the intrinsic GTPase activity of Rab3A is assayed by three methods: (1) standard assay (filter assay), (2) overlay assay, and (3) thin-layer chromatography assay. The steps used in the purification of native Rab3 GAP are: (1) preparation of the synaptic soluble fraction from rat brain, (2) QSepharose FF column chromatography, (3) hydroxyapatite column chromatography, (4) heparin-Sepharose CL-6B column chromatography, and (5) Mono Q PC 1.6/5 column chromatography. All the purification procedures are performed at 0–4°C.
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