LRRK2 microtubule sedimentation binding assay
Mariusz Matyszewski, Andrea Dickey
Abstract
Assay to determine LRRK2 protein binding to microtubules.
Original assay by Andrea Dickey. Adapted by Mariusz Matyszewski for protocols.io.
Assay originally used in Snead, Matyszewski, Dickey et al. 2022
Steps
Microtubule preparation
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 LRRK2RCKW 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 LRRK2RCKW 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.
