TITLE: In vitro kinase assay: phosphorylation of PI3Kc1 by TBK1and ULK1
AUTHORS: Elias Adriaenssens

[DESCRIPTION]
This protocol describes in vitro kinase assay, in which PI3Kc is tested for phosphorylation by TBK1 and ULK1 complex either by western blotting (S29 on ATG14L subunit) or mass spectrometry analysis.

[STEPS]
SECTION: In vitro kinase assay: phosphorylation of ATG14 by TBK1
1. Prepare Kinase Buffer without ATP and MgCl2.
SECTION: In vitro kinase assay: phosphorylation of ATG14 by TBK1
2. Prepare 100 μL of Master Mix I containing TBK1 at the final concentration of 50 nM and PI3Kc1 at the final concentration of 800 nM in the Kinase Buffer without ATP and MgCl2. 
SECTION: In vitro kinase assay: phosphorylation of ATG14 by TBK1
3. Prepare 100 μL of Master Mix II containing ULK1c at the final concentration of 200 nM and PI3Kc1 at the final concentration of 800 nM Kinase Buffer without ATP and MgCl2.
SECTION: In vitro kinase assay: phosphorylation of ATG14 by TBK1
4. Distribute 25 μL of each of the Master Mixes to new Eppendorf tubes.
SECTION: In vitro kinase assay: phosphorylation of ATG14 by TBK1
5. To start the reaction add 25 μL of Kinase Buffer with 2xATP/MgCl2 to three tubes containing the Master Mixes (time  points 5, 10 and 30 min)  and 25 μL of Kinase Buffer without ATP to one of the tubes (time point 0). To control for potential protein instability, all samples should be kept at room temperature for the same period of time. Therefore start pipetting the Kinase Buffer with 2xATP/MgCl2 to the 30 min sample, after 20 min move on to the 10 min sample and after additional 5 min to the 5 min sample, meanwhile keeping all of them in RT.
SECTION: In vitro kinase assay: phosphorylation of ATG14 by TBK1
6. To stop the reactions add 6x Protein Loading Buffer and boil the samples for 5 min at 95 °C.
SECTION: In vitro kinase assay: phosphorylation of ATG14 by TBK1
7. Separate samples via SDS-PAGE using 4-12% NuPAGE polyacrylamide gel and perform transfer (100 V for 1h at 4 °C) to a nitrocellulose membrane for western blot analysis . For Coomassie staining, incubate the SDS-PAGE gel in Coomassie stain for 10 min gently rocking at RT, destain for 10 min, and incubate in ddH2O overnight. For Mass Spectrometry analysis excise the bands the next morning with a sharp and clean scalpel.
SECTION: In vitro kinase assay: phosphorylation of ATG14 by TBK1
8. For western blot analysis continue by blocking the membrane for 1 h in Blocking Buffer gently rocking at RT.
SECTION: In vitro kinase assay: phosphorylation of ATG14 by TBK1
9. Incubate with Primary Antibodies (diluted 1:1000 in Blocking Buffer) overnight at 4 °C gently rocking.
SECTION: In vitro kinase assay: phosphorylation of ATG14 by TBK1
10. Wash the membrane 3x10 min with PBST.
SECTION: In vitro kinase assay: phosphorylation of ATG14 by TBK1
11. Incubate for 1 h with Secondary Antibodies (diluted 1:10 000 in Blocking Buffer) gently rocking at RT.
SECTION: In vitro kinase assay: phosphorylation of ATG14 by TBK1
12. Wash the membrane 3x10 min with PBST.
SECTION: In vitro kinase assay: phosphorylation of ATG14 by TBK1
13. Develop the Blot using Femto Maximum Sensitivity Substrate (ThermoFischer Scientific).