Figure S3. Workflow for membrane... | Rockefeller University Press

Figure S3.

$Workflow for membrane collection and separation for use in this study. Autophagosomes make up only a small percentage of total membrane in most cells and thus approaches to isolate autophagosome-enriched material typically require significant starting biomass. To isolate autophagosomes, we are using a density gradient centrifugation approach originally applied to rat tissue, where biomass is plentiful (Strømhaug et al., 1998), which we later adapted for use in cell culture (Jeong et al., 2009). In this illustrated workflow, we start with various cell lines grown in culture on 15-cm plates which we then scrape and Dounce homogenize before putting through a density centrifugation fractionation protocol. Our ultimate goal in this paper is to then isolate nanoscopic regions of autophagic membrane in SMA-derived nanodiscs and subject this material to various further protein–protein interrogations including a denaturant step. Each of these steps leads to a loss of material, and thus necessitates a very significant initial investment in cell culture mass. In this illustrated workflow, the numbers of plates, tubes, etc. are detailed for one replicate per cell line.$

Workflow for membrane collection and separation for use in this study. Autophagosomes make up only a small percentage of total membrane in most cells and thus approaches to isolate autophagosome-enriched material typically require significant starting biomass. To isolate autophagosomes, we are using a density gradient centrifugation approach originally applied to rat tissue, where biomass is plentiful (Strømhaug et al., 1998), which we later adapted for use in cell culture (Jeong et al., 2009). In this illustrated workflow, we start with various cell lines grown in culture on 15-cm plates which we then scrape and Dounce homogenize before putting through a density centrifugation fractionation protocol. Our ultimate goal in this paper is to then isolate nanoscopic regions of autophagic membrane in SMA-derived nanodiscs and subject this material to various further protein–protein interrogations including a denaturant step. Each of these steps leads to a loss of material, and thus necessitates a very significant initial investment in cell culture mass. In this illustrated workflow, the numbers of plates, tubes, etc. are detailed for one replicate per cell line.