Aug 25, 2023

Public workspaceIn vitro GTPase activity

DOI
dx.doi.org/10.17504/protocols.io.q26g74qwqgwz/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.q26g74qwqgwz/v1
Protocol Citation: Xinbo Wang, Pietro De Camilli 2023. In vitro GTPase activity. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g74qwqgwz/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: 68879
Keywords: GTPase, LRRK2, In vitro, ASAPCRN
Abstract
This protocol details methods for the in vitro GTPase activity testing of purified LRRK2.
Attachments
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iut9bvk9p.docx
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Materials
  • ReagentEnzChek™ Phosphate Assay KitThermo FisherCatalog #E6646
  • Reagent96-well Clear Flat Bottom Polystyrene TC-treated Microplate Individually Wrapped with Low EvaporatCorningCatalog #3595
  • Microplate reader (Synergy H1; BioTek)
In vitro GTPase activity
In vitro GTPase activity
1h 16m
1h 16m
Set up the reaction mixtures in a 96-well plate with Amount5 µL 20× reaction buffer (Concentration1 Molarity (M) Tris-HCl, Concentration20 millimolar (mM) MgCl2, Ph7.5 and Concentration2 millimolar (mM) sodium azide), Concentration200 micromolar (µM) 2-amino-6-mercapto-7-methylpurine riboside, 0.1 U purine nucleoside phosphorylase, and Concentration9 micromolar (µM) LRRK2 protein or Concentration0.8 micromolar (µM) Dynamin1 or buffers for the control in separate wells.
Note
Note: For best measurement results, we usually use a total volume of Amount80 µL -Amount100 µL .

Pipetting
Preincubate samples in the microplate reader for Duration00:30:00 at Temperature37 °C .
30m
Incubation
Add Concentration0.5 millimolar (mM) GTP (final concentration) to initiate the reaction.
Pipetting
Immediately begin reading absorbance at Thikness360 nm , every Duration00:01:00 over Duration00:45:00 at Temperature37 °C .
46m
For data analysis, subtract the last values determined before GTP was added from the corresponding values for the experimental reaction.