In vitro kinase activity

Set up the reaction mixtures with 1x kinase buffer (diluted from 10x kinase buffer), 200nanomolar (nM) LRRK2 or 8µg Rab8 protein, for the reaction group add 1millimolar (mM) ATP, and for the control add H2O instead, the total volume we used is 40µL.

Incubate samples for 2h 0m 0s at 30°C.

Quench reactions through addition of SDS sample loading buffer and heat at 95°C for 0h 10m 0s.

Resolve samples by SDS/PAGE or Phos-tag SDS/PAGE.

Detect proteins by coomassie blue staining or western blot using antibodies of Rab8 and LRRK2 phospho-specific (pT1357), respectively.