In vitro LRRK2 autophosphorylation
Pietro De Camilli, Xinbo Wang
Published: 2023-08-26 DOI: 10.17504/protocols.io.81wgb6m91lpk/v1
Abstract
This protocol details methods for the in vitro LRRK2 autophosphorylation assay.
Attachments
Steps
In vitro LRRK2 autophosphorylation
1. 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.
Set up the reaction mixture in a 1.7 mL Eppendorf tube with 1.4mL purified LRRK2 protein, 1x kinase buffer with 1millimolar (mM) ATP and 0.01U/µL GST-Prescission Protease (to remove the Flag tag).
Note
2.
Incubate samples at 4°C.
3.
Add Glutathione beads to remove GST-Prescission Protease.
4.
Concentrate samples by centrifugal filters and dialyze at 4°C against dialysis buffer.
5.
Check autophosphorylation by Western blotting using a LRRK2 phospho-specific (pT1357) antibody.
6.
Determine protein concentration by SDS-PAGE using Bovine Serum Albumin (BSA) as standard and used without freezing in the liposome tubulation experiments.
