Assay for Dual Rab GTPase binding to the LRRK2 Armadillo Domain
Suzanne R Pfeffer, Claire Y Chiang
Abstract
The LRRK2 Armadillo domain contains multiple Rab GTPase binding sites. To show that the sites can be occupied simultaneously, we use this assay. The idea is to immobilize Rab8A, bind Armadillo domain, and test if phosphoRab10 can bind to Rab8A-immobilized Armadillo domain.
Steps
Dual Rab GTPase binding to LRRK2 Armadillo Domain
Phosphorylate His-Rab10 Q68L 1-181 with His-MST3 kinase at a molar ratio of 1:3 (kinase:substrate) at 30°C 2h 0m 0s in reaction buffer. See below for details.
Pellet 50µL glutathione agarose slurry at 3000rpm,4°C.
Add GST-Rab8A Q67L to glutathione beads to achieve a concentration of 6micromolar (µM) in a total volume of 50µL reaction buffer. Incubate at 23Room temperature for 0h 30m 0s on a rotator.
Pellet beads by spinning at 3200x g and discard supernatant.
Add His-LRRK2 Armadillo domain 1-552 (10micromolar (µM) final in 50µL) or buffer alone and incubate at Room temperature for 0h 30m 0s on a rotator.
Pellet beads by spinning at 3200x g and discard supernatant.
Add phosphorylated His-Rab10 Q68L 1-181 (4micromolar (µM) in 50µL) and incubate at Room temperature for 0h 30m 0s on a rotator.
Wash beads 2X with 500µL reaction buffer.
Elute protein from beads using 50µL elution buffer (reaction buffer + 50millimolar (mM) reduced glutathione).
Pellet beads by spinning at 3200x g and collect supernatant.
Analyze eluate by SDS-PAGE and immunoblot for phosphoRab10; image blots with Li-COR, and quantify bands using ImageJ (see below for details).
