Mli 2

Titration assays were performed to determine the functional protein concentrations of the LRRK2 variants using the high-affinity inhibitor MLi-2 (Merck, U.S.A.). Therefore, 24 µl of Buffer I (25 mM Tris–HCl, 50 mM NaCl, 20 mM MgCl₂, 1 mM GDP, 1 mM DTT, 0.5 mg/ml BSA, 52.1/104.2 nM LRRK2 [total protein concentration after Bradford were mixed with 1 µl of a MLi-2 dilution series (50× concentrated) prepared in 100% DMSO [26 (link)]. The reactions were started by mixing 10 µl of this reaction mix with 10 µl of Buffer II (25 mM Tris–HCl, 50 mM NaCl, 1 mM DTT, 0.5 mg/ml BSA, 1900 µM LRRKtide, 100 µM Fluorescein-LRRKtide, 300 µM ATP) in a 384 well-plate and monitored for 60–90 min with a LabChip EZ Reader (PerkinElmer). The resulting conversion rates were plotted against the respective MLi-2 concentrations and the x-axes intersection of the respective linear fit was determined to obtain the functional protein concentrations using GraphPad Prism 6 (assuming a 1 : 1 binding of MLi-2).