Sep 23, 2022
Assay for Dual Rab GTPase binding to the LRRK2 Armadillo Domain
- Claire Chiang1,
- Suzanne Pfeffer1
- 1Department of Biochemistry, Stanford University, Stanford, CA 94305-5307
- Team Alessi
Protocol Citation: Claire Chiang, Suzanne Pfeffer 2022. Assay for Dual Rab GTPase binding to the LRRK2 Armadillo Domain. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbypzovpk/v1
Manuscript citation:
https://doi.org/10.7554/eLife.79771
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: September 23, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 70457
Keywords: ASAPCRN, lrrk2 armadillo domain the lrrk2 armadillo domain, lrrk2 armadillo domain, multiple rab gtpase, dual rab gtpase, bind armadillo domain, armadillo domain, immobilized armadillo domain, rab8a, test if phosphorab10, binding site, phosphorab10, assay, lrrk2
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: 000463
Abstract
The LRRK2 Armadillo domain contains multiple Rab GTPase binding sites. To show that the sites can be occupied simultaneously, we use this assay. The idea is to immobilize Rab8A, bind Armadillo domain, and test if phosphoRab10 can bind to Rab8A-immobilized Armadillo domain.
Materials
His-MST3 protein (pET15b 6HIS MST3 TV1; MRC-PPU DU62980)
His-Rab8A Q67L full length
His-GFP-Rab10 Q68L 1-181
Reaction buffer: 50 mM HEPES 8 , 150 mM NaCl, 5 mM MgCl2, 0.2 mM TCEP, 100 μM GTP, 2 mM ATP, 5% (v/v) glycerol
Troubleshooting
Dual Rab GTPase binding to LRRK2 Armadillo Domain
3h 36m 30s
Phosphorylate His-Rab10 Q68L 1-181 with His-MST3 kinase at a molar ratio of 1:3 (kinase:substrate) at 30 °C 02:00:00 in reaction buffer. See below for details.
Citation
LINK
2h
Pellet 50 µL glutathione agarose slurry at 3000 rpm, 4°C, 00:05:00 .
5m
Add GST-Rab8A Q67L to glutathione beads to achieve a concentration of 6 micromolar (µM) in a total volume of 50 µL reaction buffer. Incubate at Room temperature for 00:30:00 on a rotator.
30m
Pellet beads by spinning at 3200 x g, Room temperature, 00:00:30 and discard supernatant.
30s
Add His-LRRK2 Armadillo domain 1-552 (10 micromolar (µM) final in 50 µL ) or buffer alone and incubate at Room temperature for 00:30:00 on a rotator.
30m
Pellet beads by spinning at 3200 x g, 00:00:30 and discard supernatant.
30s
Add phosphorylated His-Rab10 Q68L 1-181 (4 micromolar (µM) in 50 µL ) and incubate at Room temperature for 00:30:00 on a rotator.
30m
Wash beads 2X with 500 µL reaction buffer.
Elute protein from beads using 50 µL elution buffer (reaction buffer + 50 millimolar (mM) reduced glutathione).
Pellet beads by spinning at 3200 x g, 00:00:30 and collect supernatant.
30s
Analyze eluate by SDS-PAGE and immunoblot for phosphoRab10; image blots with Li-COR, and quantify bands using ImageJ (see below for details).
Citation
LINK
Citations
Step 1
Axel Knebel, Kerryn Berndsen, Pawel Lis, Paul Davies, Dario R Alessi. Expression and purification of Rab8A (1-181) stoichiometrically phosphorylated at pThr72 (the LRRK2 site)
dx.doi.org/10.17504/protocols.io.butinwkeStep 11
Francesca Tonelli, Dario Alessi. Quantitative Immunoblotting Analysis of LRRK2 Signalling Pathway
dx.doi.org/10.17504/protocols.io.bsgrnbv6