May 23, 2022

Public workspaceLRRK2 microtubule sedimentation binding assay

DOI
dx.doi.org/10.17504/protocols.io.36wgq73b5vk5/v1
  • 1Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093;
  • 2Division of Biological Sciences, Cell and Developmental Biology Section, University of California, San Diego, La Jolla, CA 92093
Mariusz Matyszewski
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DOI: dx.doi.org/10.17504/protocols.io.36wgq73b5vk5/v1
Protocol Citation: Andrea Dickey, Mariusz Matyszewski 2022. LRRK2 microtubule sedimentation binding assay. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgq73b5vk5/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: March 03, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 59042
Keywords: LRRK2, microtubule, binding, ASAPCRN
Funders Acknowledgement:
ASAP
Grant ID: ASAP-000519
MJFF
Grant ID: 18321
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
Materials
Materials:
  • Porcine brain tubulin purchased from Cytoskeleton, Inc.
  • Purified LRRK2RCKW

Buffers:

LRRK2 Binding Buffer:
  • Concentration20 millimolar (mM) HEPES pH 7.4
  • Concentration220 millimolar (mM) NaCl
  • Concentration0.5 millimolar (mM) TCEP
  • Concentration5 % volume glycerol
  • Concentration2.5 millimolar (mM) MgCl2
  • Concentration20 micromolar (µM) GDP
  • Concentration20 micromolar (µM) Taxol

Microtubule preparation
Microtubule preparation
25m
25m
Polymerize tubulin at around Concentration2.5 mg/mL for Duration00:30:00 at Temperature37 °C . Add Taxol for stabilization and incubate for another Duration00:10:00 at Temperature37 °C .

40m
Remove free tubulin by ultracentrifugation. Centrifigation108628 x g, 37°C, 00:15:00 through a Concentration64 % volume glycerol cushion .

15m
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 (Concentration200 nanomolar (nM) in our experiments) at TemperatureRoom temperature for Duration00:10:00 with varied concentrations of microtubules in the LRRK2 binding buffer.


Note
Assay can be modified to work with other proteins. We used the same assay to monitor LRRK1RCKW binding.

10m
Pellet the microtubules by ultracentrifugation. Centrifigation108628 x g, 25°C, 00:15:00

15m
Quantify the depletion of LRRK2RCKW by taking the supernatant and boiling for Duration00:10:00 in SDS containing buffer for running a gel.

10m
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.