LRRK2 thermal shift assay
Deep Chatterjee, Sebastian Mathea, Stefan Knapp, Verena Dederer
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.
Steps
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.
