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. Author manuscript; available in PMC: 2018 Jul 5.
Published in final edited form as: Methods Mol Biol. 2015;1298:331–354. doi: 10.1007/978-1-4939-2569-8_28

Fig. 5.

Fig. 5

GSH bead-based flow cytometry assays for quantitative measurements of Rab7 guanine nucleotide binding and dissociation kinetics. (a) Assay design for detecting nucleotide binding and dissociation kinetics on Rab7 based on detection of bound fluorescent BODIPY-GTP. GST-Rab7 is immobilized on 13 μm Superdex beads coated with GSH and detection is based on fluorescent BODIPY-GTP binding. (b) BODIPY-GTP (100 nM final) was added to GST-Rab7 immobilized on GSH beads suspended in 300 μl of buffer (first arrow). The ligand was allowed to bind for 100 s and then DMSO (1 % final) or CID 1067700 (10 μM final) was added at 150 s (second arrow). While the addition of a competitive guanine nucleotide-binding inhibitor (CID 1067700) causes dissociation of BODIPY-GTP, addition of DMSO has no effect on BODIPY-GTP-binding kinetics