Subcellular Protein Fractionation Kit for Cultured Cells

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	- Instructions are followed regarding the manufacturer protocol, except fractions corresponding to cytosol, membrane and nuclei were boiled 5 min at 95°C in the presence of Laemmlli sample buffer

Protocol tips
- Instructions are followed regarding the manufacturer protocol, except fractions corresponding to cytosol, membrane and nuclei were boiled 5 min at 95°C in the presence of Laemmlli sample buffer

Publication protocol

Cells were seeded onto 10 cm2 plastic Petri dishes and cell fractionation was done according to manufacturer. Briefly, attached cells were washed three time with ice-cold PBS++ and then scraped in ice-cold PBS. Cells were transferred in microcentrifuge tubes and pelleted by centrifugation at 500 × g for 5 min. Supernatants were eliminated and cell pellets were resuspended in ice-cold cytosolic extraction buffer (CEB, Pierce). After 10 min at 4°C, tubes were centrifuged at 500 × g for 5 min. Supernatants (corresponding to cytosolic fractions) were collected. Pellets were resuspended in membrane extraction buffer (MEB, Pierce), vortexed and left 10 min at 4°C. Tubes were then centrifuged at 3000 × g for 5 min. Supernatants (corresponding to membrane fractions) were collected and pellets were resuspended in nuclear extraction buffer (NEB, Pierce), giving the nuclear fraction. Fractions corresponding to cytosol, membrane and nuclei were boiled 5 min at 95°C in the presence of Laemmlli sample buffer. Fractions were then subjected to SDS-PAGE on 10% polyacrylamide gels and transferred onto nitrocellulose membranes.

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