Aug 29, 2024

Public workspaceKinase activity assays Src and CK2

DOI
dx.doi.org/10.17504/protocols.io.5jyl82by7l2w/v1
  • 1Sascha Martens lab, University of Vienna, Max Perutz Labs - Vienna
Elias Adriaenssens
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DOI: dx.doi.org/10.17504/protocols.io.5jyl82by7l2w/v1
Protocol Citation: Elias Adriaenssens 2024. Kinase activity assays Src and CK2. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl82by7l2w/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: July 04, 2024
Last Modified: August 29, 2024
Protocol Integer ID: 103052
Keywords: ASAPCRN
Funders Acknowledgement:
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 Amount45 µL of mixes containing either only kinase
assay buffer (Concentration25 millimolar (mM) Tris-HCl Ph7.4 , Concentration150 millimolar (mM) NaCl, Concentration1 millimolar (mM) DTT, and Concentration2 millimolar (mM) MgCl2), kinase buffer and substrate
(Concentration0.5 mg/mL ) or kinase buffer, substrate (Concentration0.5 mg/mL ) and kinase (Concentration100 nanomolar (nM) ) to individual wells of a Pierce white opaque 96-well plate (Thermo Scientific).
Pipetting
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 Concentration1 micromolar (µM) .
Pipetting
Start the reactions by the addition of Amount5 µL ATP in kinase assay buffer, resulting in a final concentration of Concentration100 micromolar (µM) ATP in each of the Amount50 µL reactions.
Pipetting
After Duration01:00:00 at TemperatureRoom temperature (RT) in darkness, add Amount50 µL of Kinase-Glo Max reagent (Promega) to each well, to reach a total volume of Amount100 µL .
1h
Pipetting
Allow the luciferase reactions to stabilize for Duration00:15:00 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