Optiprep Gradient
1. Grow cells to have 3 90% confluent 15 cm plates per gradient.
2. Day 1- Prepare 25%, 20%, 15%, 10%, and 5% dilutions of Optiprep (60% iodixanol in water) in
homogenization buffer (250 mM sucrose, 10 mM Hepes-NaOH pH 7.4, 1 mM EDTA, 1 mM EGTA).
3. Layer 800 µL of each dilution from 25% to 5% in a polyallomer (13 x 51 mm) centrifuge tube. Allow
the gradient to equilibriate vertically at RT for 3 hours or overnight, covered, at 4°C.
4. Day 2- Wash cells twice with PBS- make sure to remove all PBS from plates. Harvest cells on ice in
500 µL homogenization buffer + SL inhibitors, yielding ~1 mL total homogenate.
5. Pass cells 25 times through an 18 micron clearance ball bearing homogenizer on ice.
6. Centrifuge homogenate at 800g for 10 minutes to pellet nuclei and cellular debris.
7. Carefully layer 1 mL of the supernatant onto the Optiprep gradient and centrifuge at 32.7k rpm for 3
hours at 4°C (SW50.1 rotor, Fliegel lab ultracentrifuge). Keep any extra supernatant as a total sample.
8. Collect 12 fractions of approximately 400 µL from the top of the gradient into 2 mL tubes. To
precipitate protein, add 1.2 mL of 90% acetone and leave overnight at -20°C.
9. Day 3- Centrifuge tubes at 13.2k rpm for 10 minutes at 4°C. Aspirate supernatant (acetone) and add
200 µL 100% ethanol to wash pellet. Centrifuge tubes at 13.2k rpm for 10 minutes at 4°C. Aspirate
supernatant (ethanol).
10. Resuspend pellet in 1X sample buffer (60 µL). Boil for 10 minutes, then analyze via SDS-PAGE.