Aug 25, 2023

Public workspaceFluorescence recovery after photobleaching (FRAP)

DOI
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: 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
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)
Fluorescence recovery after photobleaching (FRAP)
40m 30s
40m 30s
Prepare LRRK2-liposome mixtures in a PCR tube with Concentration300 nanomolar (nM) GFP-LRKK2, Concentration20 micromolar (µM) liposomes (labeled with trace amounts of rhodamine-PE) and Concentration1 millimolar (mM) GMPPNP.
Pipetting
PCR
Immediately depositAmount6 µL -Amount10 µL samples of step 1 on a Thikness35 mm glass bottom dish and incubate at Temperature37 °C for Duration00:30:00 .
Note
Note: Drop some buffer in the dish to prevent samples from drying out due to evaporation during incubation.

30m
Incubation
Perform FRAP experiments with a Spinning disk confocal (SDC) microscopy at TemperatureRoom temperature on a Nikon Ti-E inverted microscope using the Improvision UltraVIEW VoX system, with the settings as:
Imaging
Acquire the time-lapse images at every Duration00:00:15 .
15s
Acquire three images before bleaching.
Bleach three ROIs with a Thikness488 nm laser for 500 ms.
Acquire post-bleach images up to Duration00:10:00 at Duration00:00:15 intervals.
10m 15s