Purification of GST-Bub1-600 from Sf21 cells (large scale prep)
1. Sf21 cells were maintained in T175 flasks. You need at least 3 of these (confluent)
2. Pass 1 confluent T175 flask into 4x p150 dishes (the surface area of a T175 flask is only very slightly higher than that of a p150 dish). This way you can potentially obtain a total of 12x p150 dishes. I used only 10x p150’s.
3. Wait for 2 days so the cells are about 70-80 % confluent and ready to infect.
4. Use for each p150: 1.5 ml virus stock + 3.5 ml of complete medium. Infect for 1 hr on a rocking table (room temperature), then replace with 25 ml of complete medium.
5. Harvest and extract 42-48 hrs post infection.
6. Harvest the cells into medium using a 10 ml pipet. Transfer cells into 2x 225 ml Falcon 2075 tubes on ice. Spin 1200 rpm 10 min. The cell pellet can be stored at –80 ˚C.
7. Thaw on ice (if frozen at –80 ˚C). Extract with 2x 10 ml NETN (supplemented with protease and phosphatase inhibitors) for 20 min on rocker at 4 ˚C.
8. Transfer everything (pellet and extract) to two 30 ml centrifuge tubes and spin 10,000 rpm 10 min.
9. Transfer supernatants (pool extracts) to one 50 ml Falcon tube. Reextract pellet with 2x 10 ml NETN (supplemented with protease and phosphatase inhibitors) for 20 min on rocker at 4 ˚C
10. Pool the first extract with the second (40 ml in total). Add 1 ml GSH beads and allow binding for 1 hr at 4 ˚C with mixing.
11. Spin down and save supernatant in case binding wasn’t complete. Wash beads 4x with 50 ml NETN.
12. Elute twice with glutathione at 4˚C and save each fraction. Save beads after elution. Run SDS-PAGE to assess amount and then dialyze.