Jan 11, 2024

LRRK2 thermal shift assay V.2

DOI
https://dx.doi.org/10.17504/protocols.io.kxygx3y6kg8j/v2
  • 1Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Straße 9, Frankfurt 60438, Germany;
  • 2Structural Genomics Consortium, Buchman Institute for Molecular Life Science (BMLS), Max-von-Laue-Straße 15, Frankfurt 60438, Germany
Verena Dederer
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DOI: https://dx.doi.org/10.17504/protocols.io.kxygx3y6kg8j/v2
Protocol Citation: Verena Dederer, chatterjeedeep , Sebastian Mathea, Stefan Knapp 2024. LRRK2 thermal shift assay. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx3y6kg8j/v2
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 07, 2023
Last Modified: January 11, 2024
Protocol  Integer ID: 87480
Keywords: thermal shift assay, lrrk2 thermal shift, absorption of the fluorescent dye sypr orange, thermal shift, fluorescent dye sypr orange, protein, thermostability, scanning fluorimetry, gradual heat denaturation, lrrk2, small molecule, effect of small molecule, monitoring absorption
Funders Acknowledgements:
Aligning Science Across Parkinson’s grant
Grant ID: ASAP-000519
Abstract
Thermal shift assay or differential scanning fluorimetry analyzes the effect of small molecules on the thermostability of a protein by gradual heat denaturation and monitoring absorption of the fluorescent dye SYPR Orange at 488 nm.
Fluorescent-based thermal shift assay
Prepare 4 µM master mix of protein in buffer (20 mM Hepes pH 7.4, 150 mM NaCl, 5% glycerol) and add 1:1000 dilution of SYPR Orange.
Aliquot 20 µL of the master mix into a white 96 well plate.
Add DMSO or small molecule binder with a final concentration of 10 µM.
Seal plate, mix well and centrifuge 30 sec at 500xg.
Place plate into MX3005P real-time PCR instrument.
Measure fluorescence with excitation and emission filters set to 465 and 590 nm while gradually increase temperature 3K/min during 71 cycles from 25 to 95°C.