Purification and quantitative proteomic analysis of cell bodies and protrusions.
Ontology highlight
ABSTRACT:
SUBMITTER: Dermit M
PROVIDER: S-EPMC8065254 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:This upload concerns 2 independent but related experiments that were searched together: a) To reveal proteins that consistently localise to actin-rich protrusions across different human migratory cell-lines, we profiled the distribution of cellular proteins between protrusions and cell-bodies in a panel of 5 established human cell-lines (see experimental design). Cell lines were seeded on top of collagen-I coated 3 micron transwell filters (corning). No media was added to the bottom chamber and cells were allowed to adhere to the filter overnight. The next day, the media on top of the cells was refreshed, and media was also added to the bottom chamber in order to open the pores and allow formation of protrusions. Cells were allowed to form protrusions for 2 hrs before being fixed with ice-cold methanol. Cells were then rehydrated in PBS, followed by independent lysis of protrusions and cell-bodies on opposite sides of the filters in 2% SDS, 100mM Tris pH 7.5 b) To analyse the temporal dynamics of protein localisation to cell protrusions, we carried out a time-course spatial proteomics analysis of protrusions and cell-bodies in MDA-MB231 cells. Cells were seeded on top of collagen-I coated 3 micron transwell filters (corning). No media was added to the bottom chamber and cells were allowed to adhere to filter overnight. The next day, the media on top of cells was refreshed, and the filters were either left with no media in the bottom chamber (closed pores), or media added to the bottom chamber (open pores) for different lengths of times (1, 2, 4 , & 8 hrs) to induce protrusion formation. Cells were then fixed with ice-cold methanol, rehydrated in PBS, followed by independent lysis of protrusions and cell-bodies on opposite sides of each filter by 2% SDS, 100mM Tris pH 7.5.
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