Aug 26, 2023

In vitro GTPase activity

DOI
https://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: https://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, gtpase activity testing of purified lrrk2, gtpase activity, gtpase activity testing, purified lrrk2, gtpase activity this protocol details method, lrrk2
Abstract
This protocol details methods for the in vitro GTPase activity testing of purified LRRK2.
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iut9bvk9p.docx
16KB
Materials
  • EnzChek™ Phosphate Assay KitThermo FisherCatalog #E6646
  • 96-well Clear Flat Bottom Polystyrene TC-treated Microplate Individually Wrapped with Low EvaporatCorningCatalog #3595
  • Microplate reader (Synergy H1; BioTek)
In vitro GTPase activity
1h 16m
Set up the reaction mixtures in a 96-well plate with 5 µL 20× reaction buffer (1 Mass Percent Tris-HCl, 20 millimolar (mM) MgCl2, 7.5 and 2 millimolar (mM) sodium azide), 200 micromolar (µM) 2-amino-6-mercapto-7-methylpurine riboside, 0.1 U purine nucleoside phosphorylase, and 9 micromolar (µM) LRRK2 protein or 0.8 micromolar (µM) Dynamin1 or buffers for the control in separate wells.
Note
Note: For best measurement results, we usually use a total volume of 80 µL -100 µL .

Preincubate samples in the microplate reader for 00:30:00 at 37 °C .
30m
Add 0.5 millimolar (mM) GTP (final concentration) to initiate the reaction.
Immediately begin reading absorbance at 360 nm , every 00:01:00 over 00:45:00 at 37 °C .
46m
For data analysis, subtract the last values determined before GTP was added from the corresponding values for the experimental reaction.