Reconstitution of LRRK2 membrane recruitment onto planar lipid bilayers v1

• DOI: 10.17504/protocols.io.x54v9y7qzg3e/v1
• Publication Date: 2022-05-02T17:55:50Z
• Authors: Ayan Adhikari; Edmundo Vides; Suzanne R Pfeffer
• Keywords: 0301 basic medicine; 03 medical and health sciences

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