Large scale protein purification

1. Resuspend pellet in 40-45 mL buffer A.

2. Lyse cells using CJS lab probe sonicator for 2 mins (1s on, 2s off), keeping on ice.

(Optional: Add 100 µL protease inhibitors)

3. Transfer to 50 mL centrifuge tubes and spin down in rotor JA25.50 at 17K rpm, 1 hr, 4°C.

Meanwhile: Wash Akta pumps, lines out, and equilibrate column with buffer A.

4. Pass supernatant through 0.22µm filter using a syringe to filter out any unpelleted chunks.

5. Load sample into superloop, and proceed with Akta program as normal.