Aug 25, 2023

Public workspaceIn vitro kinase activity

DOI
dx.doi.org/10.17504/protocols.io.kxygxzr2kv8j/v1
  • 11. Departments of Neuroscience and of Cell Biology, Howard Hughes Medical Institute, Program in Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
  • 22. Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815
xinbo.wang
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DOI: dx.doi.org/10.17504/protocols.io.kxygxzr2kv8j/v1
Protocol Citation: Xinbo Wang, Pietro De Camilli 2023. In vitro kinase activity. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxzr2kv8j/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: August 19, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 68880
Keywords: Kinase activity, In vitro, LRRK2, ASAPCRN
Abstract
This protocol details methods for the in vitro kinase activity testing of purified LRRK2.
Attachments
Icon for iuuabvk9p.docx
iuuabvk9p.docx
17KB
Materials
  • ReagentSuperSep Phos-tag GelFujifilm Wako Pure ChemicalCatalog #195-1799
  • Anti-LRRK2 (phospho T1357)
  • ReagentAnti-LRRK2 (phospho T1357) antibodyAbcamCatalog #ab270606

Solutions to prepare:

10x Kinase buffer:
AB
Tris–HCl (pH 7.5)200 mM
MgCl275 mM
EGTA1 mM

In vitro kinase activity
In vitro kinase activity
2h 10m
2h 10m
Set up the reaction mixtures with 1x kinase buffer (diluted from 10x kinase buffer), Concentration200 nanomolar (nM) LRRK2 or Amount8 µg Rab8 protein, for the reaction group add Concentration1 millimolar (mM) ATP, and for the control add H2O instead, the total volume we used is Amount40 µL .
Pipetting
Incubate samples for Duration02:00:00 at Temperature30 °C .
2h
Incubation
Quench reactions through addition of SDS sample loading buffer and heat at Temperature95 °C for Duration00:10:00 .
10m
Resolve samples by SDS/PAGE or Phos-tag SDS/PAGE.
Detect proteins by coomassie blue staining or western blot using antibodies of Rab8 and LRRK2 phospho-specific (pT1357), respectively.