LRRK2RCKW single molecule kinesin motility assays
John Salogiannis
Abstract
This protocol is about LRRK2RCKW single molecule kinesin motility assays.
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.
Attachments
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 Motility Assay 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:
Prepare a 1micromolar (µM) solution of LRRK2RCKW in a cold LRRK2 buffer. Centrifuge through a 0.1 μm PVDF filter to remove aggregates. Calculate the new effective concentration. Usually around 500nanomolar (nM)-700nanomolar (nM) after centrifugation.
Create a working aliquot of LRRK2 in the desired concentration (ex. 25nanomolar (nM)-100nanomolar (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.
Next steps:
Add LRRK2RCKW sample to the channel (19µL). Incubate for 0h 5m 0s. Prepare next step while waiting.
Wash twice with the motility assay buffer supplemented with 1mg/mL.
Prepare kinesin:
Make a 4nanomolar (nM) solution of K560-GFP in the Motility Assay buffer with casein supplemented with an oxygen scavenger system (0.4%, 45µg/mL (Sigma-Aldrich), and 1.15mg/mL (Sigma-Aldrich)), 71.5millimolar (mM) and 1millimolar (mM).
Add 19µL to each chamber.
Image slide.
