May 23, 2022

Public workspaceTMR labeling of LRRK proteins

DOI
dx.doi.org/10.17504/protocols.io.ewov1nq5ogr2/v1
  • 1Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093;
  • 2Department of Biochemistry and Molecular Biology, Johns Hopkins University Bloomberg School of Public Heath, Baltimore, MD, 21205
Mariusz Matyszewski
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DOI: dx.doi.org/10.17504/protocols.io.ewov1nq5ogr2/v1
Protocol Citation: David Snead, Mariusz Matyszewski 2022. TMR labeling of LRRK proteins. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1nq5ogr2/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: March 03, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 59045
Keywords: LRRK2, LRRK1, labeling, ASAPCRN
Funders Acknowledgement:
ASAP
Grant ID: ASAP-000519
MJFF
Grant ID: 18321
Abstract
Protocol for non-specific TMR labeling of LRRK1 and LRRK2 RCKW proteins.

Protocol developed by David Snead and adapted by Mariusz Matyszewski for protocols.io.

Written as used in Snead, Matyszewski, Dickey et al. 2022.
Materials
Materials:
BODIPY TMR-X NHS Ester dye (ThermoFisher)
Purified LRRK1RCKW or LRRK2RCKW protein.
2x Micro Bio-Spin P-6 desalting columns

Buffers:
LRRK2 Storage Buffer:
  • Concentration20 millimolar (mM) HEPES pH 7.4
  • Concentration700 millimolar (mM) NaCl
  • Concentration0.5 millimolar (mM) TCEP
  • Concentration5 % volume glycerol
  • Concentration2.5 millimolar (mM) MgCl2
  • Concentration20 micromolar (µM) GDP
Typical reaction volume is Amount40 µL .
Add dye in 1:1 ratio to about Concentration20 micromolar (µM) LRRK protein .

Note
This protocol was used for LRRK1RCKW and LRRK2RCKW but should apply to other similar proteins and constructs as well.

Incubate at TemperatureRoom temperature for Duration01:00:00

1h
Remove excess dye by a buffer exchange through a Micro Bio-Spin P-6 desalting column (Bio-Rad).
Make sure to equilibrate the column with the LRRK2 Storage buffer beforehand. Follow directions included with the column.
Repeat the exchange one more time to further remove the excess dye.
Quantify protein concentration and label efficiency using a NanoDrop or equivalent method.