Crude Subcellular fractionation of FAM177A1-GFP expressing cells

Culture and transfect HeLa cells as described in dx.doi.org/10.17504/protocols.io.eq2lyp55mlx9/v1

24h 0m 0s-48h 0m 0s after transfection, wash cells in 6 well plates with PBS.

Add 200µL PBS (or fractionation buffer) to each well.

Scrape cells to release from well and transfer to a 1.7 mL eppendorph tube with additional 100µLfractionation buffer wash.

Spin the lysate at 1500rpm in a benchtop centrifuge.

Remove the supernatant and resuspend cell pellet in 1mL of cold fractionation buffer.

Homogenize resuspended cells with cell cracker (Isobiotec; 8-12 strokes), 1mL at a time.

Wash with 1mL fractionation buffer between samples.

Always use new syringes for each sample.

Ultracentrifuge lysates in a Beckman-Coulter table-top ultracentrifuge (TLA100 rotor) at 50000rpm,4°C to pellet membrane.

After the centrifugation, transfer the supernatant to a new 1.7 mL Eppendorf tube and save it for western blot analysis.

Solubilize the membrane fractions from the bottom of the tubes using 4X Laemni buffer for western blot analysis.