May 29, 2024

Public workspaceLRRK2:DARPins complex preparation

DOI
dx.doi.org/10.17504/protocols.io.j8nlkowxxv5r/v1
  • 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
Marta Sanz Murillo
Open access
DOI: dx.doi.org/10.17504/protocols.io.j8nlkowxxv5r/v1
Protocol Citation: Marta Sanz Murillo 2024. LRRK2:DARPins complex preparation. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkowxxv5r/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 10, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 86349
Keywords: ASAPCRN, cryo-EM, LRRK2, structural biology
Funders Acknowledgement:
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
LRRK2 RCKW Protein Purification
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.