In vitro GTPase activity

Set up the reaction mixtures in a 96-well plate with 5µL 20× reaction buffer (1Molarity (M) Tris-HCl, 20millimolar (mM) MgCl2, 7.5 and 2millimolar (mM) sodium azide), 200micromolar (µM) 2-amino-6-mercapto-7-methylpurine riboside, 0.1 U purine nucleoside phosphorylase, and 9micromolar (µM) LRRK2 protein or 0.8micromolar (µM) Dynamin1 or buffers for the control in separate wells.

Preincubate samples in the microplate reader for 0h 30m 0s at 37°C.

Add 0.5millimolar (mM) GTP (final concentration) to initiate the reaction.

Immediately begin reading absorbance at 360 nm , every 0h 1m 0s over 0h 45m 0s at 37°C.

For data analysis, subtract the last values determined before GTP was added from the corresponding values for the experimental reaction.