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Reconstitution of LRRK2 membrane recruitment onto planar lipid bilayers
Output Details
Description
Supported lipid bilayers have emerged as an ideal model system to study the interaction of proteins with cellular membranes. We describe here a method to monitor the recruitment of purified LRRK2 kinase onto planar lipid bilayers containing lipid-anchored Rab10 protein using Total Internal Reflection Fluorescence (TIRF) Microscopy.This method utilizes purified, FLAG-tagged, full length LRRK2 labeled with CF633 succinimidyl ester (Biotium) and bacterially expressed eGFP-Rab10-His tagged protein. LRRK2 recruitment is captured in real time at 25°C using a Nikon Ti-E inverted microscope with an Andor iXon+EMCCD camera model DU885, with PerfectFocus and a Nikon TIRF Apo 100X 1.46 NA oil immersion objective.
Identifier (DOI)
10.17504/protocols.io.x54v9y7qzg3e/v1