Liposome binding
Pietro De Camilli, Xinbo Wang
Abstract
This protocol details methods for LRRK2-liposome binding experiments analyzed by co-sedimentation or confocal fluorescence microscopy, respectively.
Attachments
iuudbvk9p.docx
Steps
Co-sedimentation analysis
Prepare samples in Beckman microfuge tubes with 300nanomolar (nM) LRRK2 or RCKW, 20micromolar (µM) PS liposomes in the absence or presence of 1millimolar (mM) GMPPNP.
Incubate samples at 37°C for 0h 30m 0s.
Centrifuge samples at 49000rpm,0h 0m 0s (100000x g,0h 0m 0s) for 0h 20m 0s in a Beckman Optima TLX ultracentrifuge.
Collect Supernatants and Pellets.
Analyze samples by SDS-PAGE and coomassie blue staining.
Confocal fluorescence microscopy analysis
For PS liposome binding. Prepare samples in PCR tubes with 20micromolar (µM) Rhod-PE labeled PS liposomes and different concentrations of LRRK2 as indicated in the main text.
For GC/PS nanotube binding. Prepare samples in PCR tubes with 20micromolar (µM) Cy5-PE labeled GC/PS nanotubes and 100nanomolar (nM) GFP-LRRK2.
Immediately deposit 6µL-10µL samples of step 6 on 35 mm glass bottom dishes and incubate at 37°C for 0h 30m 0s.
After incubation, capture the images with a spinning disk confocal (SDC) microscopy at Room temperature on a Nikon Ti-E inverted microscope using the Improvision UltraVIEW VoX system (Perkin-Elmer).
