LRRK2RCKW Widefield fluorescence microtubule binding assay

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.

Before start

Please take notice of the buffer preparation in section 'Materials'.

Make sure that you have labeled taxol-stabilized microtubules available. See the protocol here.

Steps

Create microscope slides:

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 0h 3m 0s.

Wash twice with Wash buffer .

Add a 1:150 dilution of taxol-stabilized microtubules (19µL per channel) and incubate for 0h 3m 0s .

See https://dx.doi.org/10.17504/protocols.io.bp2l6bdedgqe/v1 for making taxol-stabilized microtubules.

Wash twice with LRRK2 buffer . Add more buffer if necessary to prevent drying out.

Prepare LRRK2:

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. 25nanomolar (nM)-50nanomolar (nM)) in the LRRK2 buffer at Room temperature (recommended volume of 25µL). If adding inhibitors, add them now with DMSO. Incubate for 0h 10m 0s at Room temperature.

Adding LRRK2 and imaging:

Add LRRK2^RCKW sample to the channel (19µL). Incubate for 0h 5m 0s. Prepare next step while waiting.

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.