Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag

The overall goal of this experiment is to detect the Rab10 phosphorylation by LRRK2 using SDS-PAGE with a phosphate-binding tag. This method can help answer key questions in the Parkinson research field such as how disease mutations in LRRK2 affect Rab10 phosphorylation. The main advantage of this technique is that one can have a rough estimation of the stoichiometry of Rab10 phosphorylation using a simple western blot.

Many of the steps of this protocol are fairly standard and are described in sufficient detail by the text protocol accompanying this video. Here, we want to show you the steps that benefit most from visual instruction. Prepare plates of transfected HEK293 cells as described in the text protocol.