Sep 23, 2022

Public workspaceAssay for Dual Rab GTPase binding to the LRRK2 Armadillo Domain

DOI
dx.doi.org/10.17504/protocols.io.81wgbypzovpk/v1
  • 1Department of Biochemistry, Stanford University, Stanford, CA 94305-5307
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DOI: dx.doi.org/10.17504/protocols.io.81wgbypzovpk/v1
Protocol Citation: Claire Y Chiang, Suzanne R 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
Funders Acknowledgement:
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 Ph8 , 150 mM NaCl, 5 mM MgCl2, 0.2 mM TCEP, 100 μM GTP, 2 mM ATP, 5% (v/v) glycerol
Dual Rab GTPase binding to LRRK2 Armadillo Domain
Dual Rab GTPase binding to LRRK2 Armadillo Domain
3h 36m 30s
3h 36m 30s
Phosphorylate His-Rab10 Q68L 1-181 with His-MST3 kinase at a molar ratio of 1:3 (kinase:substrate) at Temperature30 °C Duration02:00:00 in reaction buffer. See below for details.
CITATION
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).
LINK
dx.doi.org/10.17504/protocols.io.butinwke


2h
Pellet Amount50 µL glutathione agarose slurry at Centrifigation3000 rpm, 4°C, 00:05:00 .

5m
Add GST-Rab8A Q67L to glutathione beads to achieve a concentration of Concentration6 micromolar (µM) in a total volume of Amount50 µL reaction buffer. Incubate at TemperatureRoom temperature for Duration00:30:00 on a rotator.

30m
Pellet beads by spinning at Centrifigation3200 x g, Room temperature, 00:00:30 and discard supernatant.

30s
Add His-LRRK2 Armadillo domain 1-552 (Concentration10 micromolar (µM) final in Amount50 µL ) or buffer alone and incubate at TemperatureRoom temperature for Duration00:30:00 on a rotator.

30m
Pellet beads by spinning at Centrifigation3200 x g, 00:00:30 and discard supernatant.

30s
Add phosphorylated His-Rab10 Q68L 1-181 (Concentration4 micromolar (µM) in Amount50 µL ) and incubate at TemperatureRoom temperature for Duration00:30:00 on a rotator.

30m
Wash beads 2X with Amount500 µL reaction buffer.

Elute protein from beads using Amount50 µL elution buffer (reaction buffer + Concentration50 millimolar (mM) reduced glutathione).

Pellet beads by spinning at Centrifigation3200 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
Francesca Tonelli, Dario Alessi. Quantitative Immunoblotting Analysis of LRRK2 Signalling Pathway.
LINK
dx.doi.org/10.17504/protocols.io.bsgrnbv6


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.butinwke
Step 11
Francesca Tonelli, Dario Alessi. Quantitative Immunoblotting Analysis of LRRK2 Signalling Pathway
dx.doi.org/10.17504/protocols.io.bsgrnbv6