Analysis of Plasma Membrane, Endosomal and Cytoplasmic fractions of mouse embryonic fibroblast

This project aimed to identify novel endosomal proteins and therefore cellular fractionation experiments were performed. Adherent mouse embryonic fibroblast were scraped on ice into hypotonic lysis buffer (10 mM HEPES-KOH pH 7.2, 0.25 M sucrose, 1 mM EDTA, 1 mM MgOAc and protease & phosphatase inhibitors (PhosSTOP and Complete mini tablets from Roche). Cell membranes were fragmented with French Press and nuclei removed with 10 min 1000 x g centrifugation. Plasma membrane fraction was collected with 10 min 10 000 x g centrifugation and endosomal/cytoplasmic fractions with 1h 100,000 x g centrifugation. Membrane fractions were washed at least once with lysis buffer and cytoplasmic fractions were centrifuged twice. All fractionation steps were performed at +4°C or on ice. All fractions were dissolved in sample buffer for immunoblotting