In vitro phosphorylation
Leonardo A Parra-Rivas
Published: 2023-11-12 DOI: 10.17504/protocols.io.ewov1q71ogr2/v1
Abstract
In vitro phosphorylation
Steps
1.
5 μg of α-syn GST fusion protein on beads were
resuspended in a buffer containing 20 mM HEPES, 1 mM ATP, 10 mM MgC22,
2 mM DTT, pH 7.4.
2.
The reaction was initiated by adding 1 ug of human
recombinant PLK2 (Abcam, Cat#ab102108) and incubating at 30 °C with orbital
shaking overnight (Eppendorf ThermoMixer F1.5).
3.
Samples were then analyzed by
NuPAGE and immunoblotted or alternatively analyzed by LC-MS/MS
