Nov 12, 2023
In vitro phosphorylation
- 1University of California, San Diego
Protocol Citation: Leonardo A Parra-Rivas 2023. In vitro phosphorylation. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1q71ogr2/v1
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: November 12, 2023
Last Modified: November 12, 2023
Protocol Integer ID: 90821
Abstract
In vitro phosphorylation
5 μg of α-syn GST fusion protein on beads were
resuspended in a buffer containing 20 mM HEPES, 1 mM ATP, 10 mM MgCl2,
2 mM DTT, pH 7.4.
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).
Samples were then analyzed by
NuPAGE and immunoblotted or alternatively analyzed by LC-MS/MS