Aug 25, 2023

Public workspaceIn vitro LRRK2 autophosphorylation

DOI
dx.doi.org/10.17504/protocols.io.81wgb6m91lpk/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.81wgb6m91lpk/v1
Protocol Citation: Xinbo Wang, Pietro De Camilli 2023. In vitro LRRK2 autophosphorylation. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgb6m91lpk/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: 68881
Keywords: LRRK2, Autophosphorylation, In vitro, ASAPCRN
Abstract
This protocol details methods for the in vitro LRRK2 autophosphorylation assay.
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Materials
Reagent Prescission ProteaseGenscriptCatalog #Z02799
Glutathione beads (GE Healthcare, 17075601),
ReagentAmicon Ultra-15 Centrifugal Filter Unit Millipore SigmaCatalog #UFC901024 & UFC903024 ,
ReagentSlide-A-Lyzer™ MINI Dialysis Device, 10K MWCO, 0.1 mLThermo FisherCatalog #69572 ,
ReagentAnti-LRRK2 (phospho T1357) antibodyAbcamCatalog #ab270606

Solutions to prepare:

10x Kinase buffer:
AB
Tris–HCl (pH7.5)200 mM
MgCl275 mM
EGTA1 mM
Dialysis buffer
AB
HEPES (7.4)20 mM
NaCl150 mM
MgCl22.5 mM 
Glycerol5%
DTT2 mM
GDP20 μM
In vitro LRRK2 autophosphorylation
In vitro LRRK2 autophosphorylation
Set up the reaction mixture in a 1.7 mL Eppendorf tube with Amount1.4 mL purified LRRK2 protein, 1x kinase buffer with Concentration1 millimolar (mM) ATP and Amount0.01 U/µL GST-Prescission Protease (to remove the Flag tag).
Note
Note: the LRRK2 protein used in this experiment was obtained by elution from the anti-FLAG M2 resin as described in the LRRK2 purification protocol.


Pipetting
Incubate samples DurationOvernight at Temperature4 °C .
Incubation
Overnight
Add Glutathione beads to remove GST-Prescission Protease.
Pipetting
Concentrate samples by centrifugal filters and dialyze DurationOvernight at Temperature4 °C against dialysis buffer.
Overnight
Check autophosphorylation by Western blotting using a LRRK2 phospho-specific (pT1357) antibody.
Determine protein concentration by SDS-PAGE using Bovine Serum Albumin (BSA) as standard and used without freezing in the liposome tubulation experiments.