[32P]GTPgS filter binding assay.
Dynamin (approximately 2uM) was incubated with 100uM GTPgS spiked with [32P]GTPgS (approximately 10uCi/ml) in GTPase assay buffer (see main paper materials and methods) for 20min. at room temperature. Sample volumes were 50ul. After incubation the samples were applied to a sheet of nitrocellulose in a slot-blotter, under vacuum. The slots to which samples had been applied were then washed with 3 applications of 150ul of ice cold GTPase assay buffer. After washing, the nitrocellulose was removed from the blotter, left to dry in a 37oC air oven and then placed in a phosphoimager cassette. Analysis was carried out using a Molecular Dynamics phosphoimager and ImageQuant software.
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