Induction of non-selective bulk autophagy
Elias Adriaenssens
Published: 2023-09-23 DOI: 10.17504/protocols.io.4r3l228b3l1y/v1
Abstract
This protocol describes how to induce bulk (non-selective) autophagy in HeLa cells through nutrient starvation.
Attachments
Steps
Nutrient starvation experiments
1.
To induce bulk autophagy, starve the cells by culturing them in Hank balanced salt medium
(HBSS, Thermo Fisher) for the indicated time.
2.
Collect cells by trypsinization, wash them in PBS and lyse them in RIPA buffer for 0h 20m 0s on ice. Clear the samples by centrifugation at 20000x g,4°C.
3.
Collect the soluble supernatant fraction and measure the protein concentrations using the Pierce Detergent Compatible Bradford Assay Kit (23246, Thermo Fisher).
4.
Analyse protein samples by SDS-PAGE and western blot analysis.
