Sep 23, 2023
Induction of non-selective bulk autophagy
- Elias Adriaenssens1
- 1Sascha Martens lab, University of Vienna, Max Perutz Labs - Vienna
External link: https://doi.org/10.1038/s41594-024-01338-y
Protocol Citation: Elias Adriaenssens 2023. Induction of non-selective bulk autophagy . protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l228b3l1y/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: July 04, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 84443
Keywords: Nutrient starvation experiments, ASAPCRN, autophagy in hela cell, selective autophagy, autophagy, hela cell, nutrient starvation, cell, bulk
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-000350
Marie Skłodowska-Curie MSCA Postdoctoral fellowship
Grant ID: 101062916
Abstract
This protocol describes how to induce bulk (non-selective) autophagy in HeLa cells through nutrient starvation.
Attachments
773-1958.pdf
29KB
Materials
Reagents
- Hank balanced salt medium (HBSS, Thermo Fisher).
- Pierce™ Detergent Compatible Bradford Assay KitThermo FisherCatalog #23246
RIPA buffer
| A | B | |
| Tris-HCl pH 8.0 | 50 mM | |
| NaCl | 150 mM | |
| sodium deoxycholate | 0.50% | |
| SDS | 0.10% | |
| NP-40 | 1% | |
| cOmplete mini-EDTA protease inhibitor | ||
| Roche Phosstop |
Troubleshooting
Nutrient starvation experiments
30m
To induce bulk autophagy, starve the cells by culturing them in Hank balanced salt medium
(HBSS, Thermo Fisher) for the indicated time.
Collect cells by trypsinization, wash them in PBS and lyse them in RIPA buffer for 00:20:00 on ice. Clear the samples by centrifugation at 20000 x g, 4°C, 00:10:00 .
30m
Collect the soluble supernatant fraction and measure the protein concentrations using the Pierce Detergent Compatible Bradford Assay Kit (23246, Thermo Fisher).
Analyse protein samples by SDS-PAGE and western blot analysis.