Sep 13, 2024
Version 2
LRRK2 PhosphoSens Assay V.2
- Nicolai D. Raig1,2,
- Stefan Knapp1,2
- 1Structural Genomics Consortium, Buchman Institute for Molecular Life Science (BMLS), Max-von-Laue-Straße 15, Frankfurt 60438, Germany;
- 2Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Straße 9, Frankfurt 60438, Germany
Protocol Citation: Nicolai D. Raig, Stefan Knapp 2024. LRRK2 PhosphoSens Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbz8qngpk/v2Version created by Nicolai D. Raig
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: September 12, 2024
Last Modified: September 13, 2024
Protocol Integer ID: 107565
Funders Acknowledgement:
Aligning Science Across Parkinson’s
Grant ID: ASAP-000519
Abstract
With this enzymatic activity assay we were able to determine IC50 values of published inhibitors aswell as newly synthesized compounds that inhibit LRRK2. The assay is based on the in vitro phosphorylation reaction between the enzymatic active LRRK2(RCKW)-construct and the SOX-based substrate peptide (AQT0615 from AssayQuant Technologies).
Make 10mM, 100µM and 1.6µM stock solutions of the compounds in DMSO.
1h
Pipett a dilution series of eleven concentrations between 15µM and 0.4nM (calculated with an assay volume of 10µL) of the compounds into white 384-well plates (Greiner 781207) as duplicates with an ECHO acoustic dispenser (Labcyte). Pipett a equivalent of DMSO in two wells per compound as 0% (without protein and compound) and 100%(without compound) control.
20m
Dilute the purified LRRK2RCKW to 22nM with the reaction buffer (50mM HEPES buffer (pH7.5), 10mM MgCl2, 1% glycerol, 1mM DTT, 0.2mg/mL BSA, 0.01% Tween20, 5µM AQT0615) and add 10µL to each well, except the 0% control wells, with a multichannel pipette (Eppendorf). Add 10µL of pure reaction buffer to the 0% control wells.
20m
Pipett 5nL 100mM ATP stock into each well with the ECHO acoustic dispenser (Labcyte).
10m
Centrifuge the plates at 1500gn for 2min, before incubating the reaction at room temperature for 4h.
4h
Measure the fluorescence after 360nm excitation at 487nm emission with a PHERAstar plate reader (BMG Labtech).
10m
Normalize the response to the average 0% and 100% controls. Calculate the IC50 values via non-linear regression of the log[inhibitor] vs. the normalized response with GraphPad Prism 8.
30m