Aug 29, 2024
Kinase activity assays Src and CK2
- 1Sascha Martens lab, University of Vienna, Max Perutz Labs - Vienna
External link: https://doi.org/10.1038/s41556-025-01712-y
Protocol Citation: Elias Adriaenssens 2024. Kinase activity assays Src and CK2. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl82by7l2w/v1
Manuscript citation:
Adriaenssens, E., Schaar, S., Cook, A.S.I. et al. Reconstitution of BNIP3/NIX-mitophagy initiation reveals hierarchical flexibility of the autophagy machinery. Nat Cell Biol 27, 1272–1287 (2025). https://doi.org/10.1038/s41556-025-01712-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: July 04, 2024
Last Modified: August 29, 2024
Protocol Integer ID: 103052
Keywords: ASAPCRN, protocol details about the kinase activity assay, kinase activity assay, kinase activity, ck2, src
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-000350
Marie Skłodowska-Curie MSCA Postdoctoral fellowship
Grant ID: 101062916
Abstract
This protocol details about the Kinase activity assays of Src and CK2.
To verify the activity of kinases SRC and CK2, add 45 µL of mixes containing either only kinase
assay buffer (25 millimolar (mM) Tris-HCl 7.4 , 150 millimolar (mM) NaCl, 1 millimolar (mM) DTT, and 2 millimolar (mM) MgCl2), kinase buffer and substrate
(0.5 mg/mL ) or kinase buffer, substrate (0.5 mg/mL ) and kinase (100 nanomolar (nM) ) to individual wells of a Pierce white opaque 96-well plate (Thermo Scientific).
Substrate peptides used were RRRDDDSDDD 10-mer (PEP-CK2I-025, Biaffin) and Poly-(Glu,Tyr 4:1) (40217, BPS) for CK2 and Src kinases, respectively. For CK2, add a specific inhibitor Silmitasertib CX-4945 (S2248, Selleck- chem), where indicated, at a concentration of 1 micromolar (µM) .
Start the reactions by the addition of 5 µL ATP in kinase assay buffer, resulting in a final concentration of 100 micromolar (µM) ATP in each of the 50 µL reactions.
After 01:00:00 at Room temperature (RT) in darkness, add 50 µL of Kinase-Glo Max reagent (Promega) to each well, to reach a total volume of 100 µL .
1h
Allow the luciferase reactions to stabilize for 00:15:00 before measuring luciferase activity at a Spark Multi-Mode Microplate Reader (TECAN). The luciferase activity correlates with ATP quantity, and thus, an inverse relationship between measured luminescence and kinase activity exists.
15m