Liposome tubulation
Pietro De Camilli, Xinbo Wang
Abstract
This protocol details methods for the LRRK2-induced liposome tubulation experiment and its analysis by confocal fluorescence microscopy and negative stained electron microscopy.
Attachments
iuuebvk9p.docx
Steps
Confocal fluorescence microscopy analysis
Prepare the samples in a PCR tube with 300nanomolar (nM) LRKK2 proteins (WT or mutant full length LRRK2 or RCKW), 20micromolar (µM) liposomes with or without 1millimolar (mM) GMPPNP (or other guanylnucleotides).
Immediately deposit 6µL -10µL samples of step 1 on a 35 mm glass bottom dish and incubate at 37°C for 0h 30m 0s.
After incubation, capture 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).
Negative stained electron microscopy (EM) analysis
Glow-discharge carbon-coated grids (25 mA, 0h 0m 45s).
Place the discharged grids into a 35 mm glass bottom dish.
Prepare samples in a PCR tube with 300nanomolar (nM) LRKK2, 80micromolar (µM) liposomes and 1millimolar (mM) GMPPNP.
Immediately apply 6µL of the mixture to the grid and incubate the mixture at 37°C for 0h 30m 0s.
Blot the grid with filter paper after incubation and stain samples with 2% uranyl acetate for 0h 0m 40s.
Dry the grid with filter paper.
Take images using a Talos L 120C TEM microscope at 80 kV with Velox software and a 4k × 4K Ceta CMOS Camera (Thermo Fisher Scientific).
