Aug 25, 2023

Fluorescence recovery after photobleaching (FRAP)

DOI
https://dx.doi.org/10.17504/protocols.io.5qpvobj29l4o/v1
  • 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
xinbo.wang
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DOI: https://dx.doi.org/10.17504/protocols.io.5qpvobj29l4o/v1
Protocol Citation: Xinbo Wang, Pietro De Camilli 2023. Fluorescence recovery after photobleaching (FRAP). protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvobj29l4o/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: 68888
Keywords: Photobleaching, Fluorescence, LRRK2, ASAPCRN, fluorescence recovery, induced liposome tubule, liposome tubule, liposome, analysis of lrrk2
Abstract
This protocol details methods of the FRAP analysis of LRRK2-induced liposome tubules in vitro
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Fluorescence recovery after photobleaching (FRAP)
40m 30s
Prepare LRRK2-liposome mixtures in a PCR tube with 300 nanomolar (nM) GFP-LRKK2, 20 micromolar (µM) liposomes (labeled with trace amounts of rhodamine-PE) and 1 millimolar (mM) GMPPNP.
Immediately deposit6 µL -10 µL samples of step 1 on a 35 mm glass bottom dish 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
Perform FRAP experiments with a Spinning disk confocal (SDC) microscopy at Room temperature on a Nikon Ti-E inverted microscope using the Improvision UltraVIEW VoX system, with the settings as:
Acquire the time-lapse images at every 00:00:15 .
15s
Acquire three images before bleaching.
Bleach three ROIs with a 488 nm laser for 500 ms.
Acquire post-bleach images up to 00:10:00 at 00:00:15 intervals.
10m 15s