May 23, 2022
LRRK2RCKW Widefield fluorescence microtubule binding assay
- David M. Snead1,2
- 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 Health, Baltimore, MD, 21205
Protocol Citation: David M. Snead 2022. LRRK2RCKW Widefield fluorescence microtubule binding assay. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxz7bdv8j/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: April 18, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 60956
Keywords: LRRK2, imaging, ASAPCRN, microtubule, binding
Funders Acknowledgement:
ASAP
Grant ID: ASAP-000519
MJFF
Grant ID: 18321
Abstract
This assay uses TMR labeled LRRK2 or LRRK1 RCKW to measure binding to microtubules in vitro.
Created by David Snead. Adapted to protocols.io by Mariusz Matyszewski.
As used in Snead, Matyszewski, Dickey et al. 2022.
Guidelines
Similar setup as in:
CITATION
Also see:
Image analysis:
Image analysis was performed with ImageJ. Average TMR-LRRK2RCKW fluorescence intensity per microtubule was calculated from a 1 pixel-wide line drawn along the long axis of the microtubule; overall average background fluorescence intensity was subtracted. These background-subtracted intensities were averaged over all microtubules per field of view, normalized by microtubule length, to yield a single data point. Eight fields of view at each concentration of LRRK2RKCW were then averaged.
Materials
Recommended Equipment and Setup:
This single-molecule imaging experiment was originally performed using total internal reflection fluorescence (TIRF) microscopy with an inverted microscope (Nikon, Ti-E Eclipse) equipped with a 100x 1.49 N.A. oil immersion objective (Nikon, Plano Apo), and a MLC400B laser launch (Agilent), with 405 nm, 488 nm, 561 nm and 640 nm laser lines. Excitation and emission paths were filtered using single bandpass filter cubes (Chroma), and emitted signals were detected with an electron multiplying CCD camera (Andor Technology, iXon Ultra 888). Illumination and image acquisition were controlled with NIS Elements Advanced Research software (Nikon), and the xy position of the stage was controlled with a ProScan linear motor stage controller (Prior).
Required Buffers:
Streptavidin Buffer:
- 0.5 mg/mL Streptavidin
- 30 millimolar (mM) HEPES pH 7.4
- 2 millimolar (mM) MgOAc
- 1 millimolar (mM) EGTA
- 10 % Glycerol
Wash Buffer:
- 30 millimolar (mM) HEPES pH 7.4
- 50 millimolar (mM) KOAc
- 2 millimolar (mM) MgOAc
- 1 millimolar (mM) EGTA
- 10 % Glycerol
- 1 millimolar (mM) DTT
- 200 micromolar (µM) Taxol
LRRK2 Buffer:
- 20 millimolar (mM) HEPES pH 7.4
- 80 millimolar (mM) NaCl
- 0.5 millimolar (mM) TCEP
- 5 % Glycerol
- 0.5 millimolar (mM) MgCl2
- 20 micromolar (µM) GDP
Safety warnings
For hazard information and safety warnings, please refer to the SDS (Safety Data Sheet).
Before start
Please take notice of the buffer preparation in section 'Materials'.
Create microscope slides:
Create microscope slides:
1h 11m
1h 11m
Adhere Biotin-PEG-functionalized coverslips (Microsurfaces) to a microscope slide using double-sided scotch tape, creating 4 channels per slide.
Add the streptavidin buffer to each channel and incubate for 00:03:00 .
3m
Wash twice with Wash buffer.
Add a 1:150 dilution of taxol-stabilized microtubules (19 µL per channel) and incubate for 00:03:00 .
See https://dx.doi.org/10.17504/protocols.io.bp2l6bdedgqe/v1 for making taxol-stabilized microtubules.
3m
Wash twice with LRRK2 buffer. Add more buffer if necessary to prevent drying out.
Prepare LRRK2:
Prepare LRRK2:
1h 11m
1h 11m
Make sure to use TMR labelled protein. See https://dx.doi.org/10.17504/protocols.io.ewov1nq5ogr2/v1 for labeling protocol.
Create a working aliquot of LRRK2 (or LRRK1) in the desired concentration (ex. 25 nanomolar (nM) -50 nanomolar (nM) ) in the LRRK2 buffer at Room temperature (recommended volume of 25 µL ). If adding inhibitors, add them now with DMSO. Incubate for 00:10:00 at Room temperature .
10m
Adding LRRK2 and imaging:
Adding LRRK2 and imaging:
5m
5m
Add LRRK2RCKW sample to the channel (19 µL ). Incubate for 00:05:00 . Prepare next step while waiting.
5m
Image slide. We imaged using multiple fields of view along the flow chamber with the objective in widefield illumination, with successive excitation at 488 nm (15% laser power, 100 ms exposure) and 561 nm (25% laser power, 100 ms exposure).
Check guidelines for image analysis notes.
Citations
Deniston CK, Salogiannis J, Mathea S, Snead DM, Lahiri I, Matyszewski M, Donosa O, Watanabe R, Böhning J, Shiau AK, Knapp S, Villa E, Reck-Peterson SL, Leschziner AE. Structure of LRRK2 in Parkinson's disease and model for microtubule interaction.
https://doi.org/10.1038/s41586-020-2673-2