Sep 23, 2023

Public workspaceIn vitro kinase assay

Nature Structural & Molecular Biology
DOI
https://dx.doi.org/10.17504/protocols.io.4r3l225xjl1y/v1
  • Elias Adriaenssens1
  • 1Sascha Martens lab, University of Vienna, Max Perutz Labs - Vienna
Elias Adriaenssens
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DOI: https://dx.doi.org/10.17504/protocols.io.4r3l225xjl1y/v1
External link: https://doi.org/10.1038/s41594-024-01338-y
Protocol Citation: Elias Adriaenssens 2023. In vitro kinase assay. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l225xjl1y/v1
Manuscript citation:
Elias Adriaenssens, Thanh Ngoc Nguyen, Justyna Sawa-Makarska, Grace Khuu, Martina Schuschnig, Stephen Shoebridge, Marvin Skulsuppaisarn, Emily Maria Watts, Kitti Dora Csalyi, Benjamin Scott Padman, Michael Lazarou, Sascha Martens (2024) Control of mitophagy initiation and progression by the TBK1 adaptors NAP1 and SINTBAD.Nature Structural & Molecular Biology doi: 10.1038/s41594-024-01338-y
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: June 27, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 84091
Keywords: in vitro kinase assay, ASAPCRN, kinase, assay this protocol, assay
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-000350
Abstract
This protocol describes in vitro kinase assay.
Attachments
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Materials
Materials

  • Recombinant proteins TBK1, ULK1 complex, and NAP1
  • MgCl2
  • ATP
  • dH2O
  • Nitrocellulose membranes (RPN132D, GE Healthcare)
  • Mini Trans-Blot Cell (Bio-Rad).
  • SDS-PAGE gels (NP0321BOX, NP0322BOX, or NP0323BOX, Thermo Fisher)
  • PageRuler Prestained protein marker (Thermo Fisher)
Kinase buffer
AB
Tris-HCl pH 7.420 mM
NaCl150 mM
DTT1  mM
Fixation solution
A
40% ethanol
10% acetic acid
50% dH2O
In vitro kinase assay
25m
Mix recombinant proteins TBK1 or ULK1-complex (composed of ULK1, FIP200, ATG13, and ATG101) and NAP1 in kinase buffer.
Mix
Use the kinases at Concentration50 nanomolar (nM) and mix with Concentration250 nanomolar (nM) NAP1.

Start the kinase reactions by the adding 2x ATP/MgCl2 kinase buffer to a final concentration of Concentration10 millimolar (mM) MgCl2 and Concentration100 millimolar (mM) ATP.

Prepare protein mixtures as master mixes and divide over the number of time points.
To control for potential protein instability, induce the latest time point first and then go gradually to the shortest time point.
In this way, keep all protein mixtures at TemperatureRoom temperature for the same time, and terminate the reactions together.
Achieve the termination of reactions by the addition of 6x Protein Loading dye and heat inactivation at Temperature95 °C for Duration00:05:00 .

5m
Temperature
Separate the samples on 4-12% SDS-PAGE gels (NP0321BOX, NP0322BOX, or NP0323BOX, Thermo Fisher) with PageRuler Prestained protein marker (Thermo Fisher).
After the run, either stain the SDS-PAGE gel with Coomassie or transfer to nitrocellulose membranes for western blot analysis.
In the case of Coomassie staining, incubate the gel for Duration00:10:00 in Coomassie solution, fix for Duration00:10:00 with fixation solution, and then destain it DurationOvernight in dH2O.
30m
Incubation
Overnight
Cut the band corresponding to NAP1 from the gel with a fresh scalpel and submit for mass spectrometry analysis.
In the case of western blotting, transfer the proteins onto nitrocellulose membranes (RPN132D, GE Healthcare) for Duration01:00:00 at Temperature4 °C using the Mini Trans-Blot Cell (Bio-Rad).
1h
Process the membranes further for western blot analysis, as described in the western blot protocol.