May 29, 2024
LRRK2:DARPins complex preparation
- 1Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093, USA;
- 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA
External link: https://dx.doi.org/10.1016/j.jbc.2024.107469
Protocol Citation: Marta Sanz Murillo 2024. LRRK2:DARPins complex preparation. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkowxxv5r/v1
Manuscript citation:
Dederer V, Murillo MS, Karasmanis EP, Hatch KS, Chatterjee D, Preuss F, Azeez KRA, Nguyen LV, Galicia C, Dreier B, Plückthun A, Versees W, Mathea S, Leschziner AE, Reck-Peterson SL, Knapp S (2024) A designed ankyrin-repeat protein that targets Parkinson’s disease-associated LRRK2. The Journal of Biological Chemistry 300(7). doi: 10.1016/j.jbc.2024.107469
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 11, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 86349
Keywords: ASAPCRN, cryo-EM, LRRK2, structural biology
Funders Acknowledgements:
Aligning Science Across Parkinson's: ASAP
Grant ID: ASAP-000519
Abstract
Protocol used to create LRRK2-RCKW: DARPin complex for cryo-EM grid preparation.
LRRK2:DARPins complex preparation
LRRK2:DARPins complex preparation
His6-Z-TEV-LRRK2-RCKW was expressed and purified as described in a previous protocol
Protocol
NAME
LRRK2 RCKW Protein Purification
CREATED BY
Mariusz Matyszewski
Prepare LRRK2 buffer exchange: Keep it at 4ºC.
20 millimolar (mM) HEPES pH=7.4
150 millimolar (mM) NaCl
2.5 millimolar (mM) MgCl2
20 micromolar (µM) GDP
0.5 millimolar (mM) TCEP
Spin down purified LRRK2-RCKW (10000 rcf, 4°C, 10 minutes). Leave protein on ice afterward.
Note
For the best result, keep protein on ice and reduce the amount of time between spinning
and freezing cryo-EM samples.
Exchange buffer using a spin desalting column (Zeba™ Spin Desalting Columns, 7K MWCO (Catalog number: 89877).
Spin down again the exchange buffer LRRK2-RCKW (10000 rcf, 4°C, 10 minutes) and measure the concentration. Leave protein on ice afterward
Thaw the DARPin protein of your interest (E11 or C12) and spin it down. Measure its concentration.
Based on LRRK2-RCKW concentration, add the necessary volume to get a proportional ratio
LRRK2:DARPin 1:1.25 and dilute to a final 10 micromolar (µM) LRRK2-RCKW concentration using exchange buffer (150 mM NaCl)
Incubate 10 minutes at RT. Afterward, keep it on ice until grid preparation.