Aug 25, 2023
Liposome binding
- Xinbo Wang1,2,
- Pietro De Camilli1,2
- 11. Departments of Neuroscience and of Cell Biology, Howard Hughes Medical Institute, Program in Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
- 22. Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815
Protocol Citation: Xinbo Wang, Pietro De Camilli 2023. Liposome binding. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvmndqng3p/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 19, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 68884
Keywords: Liposome binding, Co-sedimentation, Fluorescence microscopy, ASAPCRN
Abstract
This protocol details methods for LRRK2-liposome binding experiments analyzed by co-sedimentation or confocal fluorescence microscopy, respectively.
Attachments
iuudbvk9p.docx
19KB
Co-sedimentation analysis
Co-sedimentation analysis
Prepare samples in Beckman microfuge tubes with 300 nanomolar (nM) LRRK2 or RCKW, 20 micromolar (µM) PS liposomes in the absence or presence of 1 millimolar (mM) GMPPNP.
Incubate samples at 37 °C for 00:30:00 .
30m
Centrifuge samples at 49000 rpm (100000 x g ) for 00:20:00 in a Beckman Optima TLX ultracentrifuge.
20m
Collect Supernatants and Pellets.
Note
Note: Pellets were resuspended with the same volume of protein buffer as the supernatant.
Analyze samples by SDS-PAGE and coomassie blue staining.
Confocal fluorescence microscopy analysis
Confocal fluorescence microscopy analysis
30m
30m
For PS liposome binding. Prepare samples in PCR tubes with 20 micromolar (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 20 micromolar (µM) Cy5-PE labeled GC/PS nanotubes and 100 nanomolar (nM) GFP-LRRK2.
Immediately deposit 6 µL -10 µL samples of step 6 on 35 mm glass bottom dishes and incubate at 37 °C for 00:30:00 .
Note
Note: Drop some buffer in the dish to prevent samples from drying out due to evaporation during incubation.
30m
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).