LRRK2 microtubule sedimentation binding assay

Polymerize tubulin at around 2.5mg/mL for 0h 30m 0s at 37°C . Add Taxol for stabilization and incubate for another 0h 10m 0s at 37°C .

Remove free tubulin by ultracentrifugation. 108628x g,37°C through a 64% volume .

Resuspend the resulting microtubule pellet in the LRRK2 binding buffer .

Determine microtubule concentration by running an SDS-PAGE with actin standards.

Incubate desired amount of LRRK2^RCKW protein (200nanomolar (nM) in our experiments) at Room temperature for 0h 10m 0s with varied concentrations of microtubules in the LRRK2 binding buffer .

Pellet the microtubules by ultracentrifugation. 108628x g,25°C

Quantify the depletion of LRRK2^RCKW by taking the supernatant and boiling for 0h 10m 0s in SDS containing buffer for running a gel.

Samples were run on 4-12% polyacrylamide gels (NuPage, Invitrogen) and stained with SYPRO-Red Protein Gel Stain (ThermoFisher) for protein detection.

Binding curves were fit in GraphPad Prism (9.2; GraphPad Software) with a nonlinear regression hyperbolic curve.