In vitro LRRK2 kinase activity assay using mass-spectrometry as readout
Sebastian Mathea, Stefan Knapp, Verena Dederer, chatterjeedeep
Abstract
This protocol can be used in this form or with small adjustments regarding concentration and reaction time to determine in vitro substrate phosphorylation for any purified kinase substrate pair.
It can be used to determine inhibition curves by addition of concentration series of kinase inhibitors.
Reaction is carried out in 50 µL reaction mix containing final concentration of 50 nM kinase and 5 mM substrate.
Steps
Step-by-Step Protocol
Prepare 50 µL reaction mix I: 50 nM purified LRRK2 and 5 µM Rab8 substrate in buffer containing 20 mM Hepes pH 7.4, 150 mM NaCl, 5% glycerol, 0.5 mM TCEP, 20 µM GDP, 2.5 mM MgCl2.
Aliquot 25 µL per tube.
Prepare 25 µL reaction mix II: 2 mM ATP + 2.5 mM MgCl2 in buffer containing 20 mM Hepes pH 7.4, 150 mM NaCl, 5% glycerol, 0.5 mM TCEP, 20 µM GDP, 2.5 mM MgCl2.
Prepare 25 µL reaction mix III: same as reaction mix II but no ATP as negative control.
Start reaction by adding 25 µL reaction II to reaction mix I.
Note: for negative control add 25 µL reaction mix III to second aliquot of reaction mix I.
Mix by vortexing and brief centrifugation for 30 sec with 500xg.
Incubate reaction for 3 h at room temperature.
Note: depeding on kinase this may be shorter or longer.
Stopp reaction by adding 50 µL mass spec buffer (dH2O+0.1% formic acid).
Store at -80°C or proceed directly with mass spectrometry analysis.
