Fluorescence recovery after photobleaching (FRAP)

Prepare LRRK2-liposome mixtures in a PCR tube with 300nanomolar (nM) GFP-LRKK2, 20micromolar (µM) liposomes (labeled with trace amounts of rhodamine-PE) and 1millimolar (mM) GMPPNP.

Immediately deposit6µL -10µL samples of step 1 on a 35 mm glass bottom dish and incubate at 37°C for 0h 30m 0s.

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 0h 0m 15s.

Acquire three images before bleaching.

Bleach three ROIs with a 488 nm laser for 500 ms.

Acquire post-bleach images up to 0h 10m 0s at 0h 0m 15s intervals.