TITLE: Purification of human ATP13A2 for cryo-EM analysis
AUTHORS: Sue  Sim, eunyong_park  

[DESCRIPTION]
Purification of GFP-tagged human ATP13A2 expressed in Sf9 cells for cryo-EM analysis

[STEPS]
SECTION: Day 1: Crude membrane preparation and solubilization
1. Thaw Sf9 cell pellets at room temperature (typical size around 10 g from 0.7L of culture)
SECTION: Day 1: Crude membrane preparation and solubilization
2. All subsequent steps performed at 4 °C 

SECTION: Day 1: Crude membrane preparation and solubilization
3. Resuspend each pellet in 30 mL Lysis Buffer (use 3x volume of cell pellet, 40 mL total volume)
SECTION: Day 1: Crude membrane preparation and solubilization
4. Homogenize pellet with Dounce homogenizer, 100 plunges tight on ice
SECTION: Day 1: Crude membrane preparation and solubilization
5. Pour lysate into pre-chilled 50 mL centrifuge tubes
SECTION: Day 1: Crude membrane preparation and solubilization
6. Spin in centrifuge at 4000 x g, 10 min, 4 °Cto remove unbroken cells

SECTION: Day 1: Crude membrane preparation and solubilization
7. Transfer supernatant to pre-chilled ultracentrifuge rotor tubes
SECTION: Day 1: Crude membrane preparation and solubilization
8. Spin lysate in ultracentrifuge at 100000 x g, 90 min, 4 °C to pellet membranes (Beckman Type 45 Ti rotor)

SECTION: Day 1: Crude membrane preparation and solubilization
9. Resuspend membrane pellet in Lysis Buffer and final volume 1% DDM/0.2% CHS (1X pellet, 7X Lysis Buffer, 2X 5% DDM/1% CHS)
    DDM: n-dodecyl-β-D-maltopyranoside (Anatrace)
    CHS: cholesteryl hemisuccinate (Anatrace)
SECTION: Day 1: Crude membrane preparation and solubilization
10. Solubilize by rotating end-over-end for 150 min at 4 °C 

SECTION: Day 1: Crude membrane preparation and solubilization
11. Clarify lysate in ultracentrifuge at 100000 x g, 60 min, 4 °C 

SECTION: Day 1: Bead binding and overnight 3C cleavage
12. Equilibrate 1 mL Sepharose beads conjugated with anti-GFP nanobody with Wash Buffer
SECTION: Day 1: Bead binding and overnight 3C cleavage
13. Add beads to gravity column and wash with 10 mL wash buffer
SECTION: Day 1: Bead binding and overnight 3C cleavage
14. After ultracentrifugation is complete, transfer supernatant into 50 mL falcon tube
SECTION: Day 1: Bead binding and overnight 3C cleavage
15. Add 1 mL equilibrated anti-GFP nanobody beads to tube
SECTION: Day 1: Bead binding and overnight 3C cleavage
16. Incubate by rotating end-over-end for 150 min at 4 °C 

SECTION: Day 1: Bead binding and overnight 3C cleavage
17. Transfer to gravity column and let flow-through drain
SECTION: Day 1: Bead binding and overnight 3C cleavage
18. Wash beads with 30 column volumes of Wash Buffer
SECTION: Day 1: Bead binding and overnight 3C cleavage
19. Add 5 mL Wash Buffer to gravity column and 10 µg/mL HRV 3C protease
SECTION: Day 1: Bead binding and overnight 3C cleavage
20. Incubate by rotating end-over-end overnight at 4 °C 

SECTION: Day 2: SEC column
21. Equilibrate Superose 6 Increase 10/300 GL column with Wash Buffer
SECTION: Day 2: SEC column
22. Concentrate the protein to 0.5 mL using an Amicon Ultrafilter (cut-off 100kDa)
SECTION: Day 2: SEC column
23. After concentration, spin protein at 17000 x g, 10 min, 4 °C 

SECTION: Day 2: SEC column
24. Injected sample into FPLC
SECTION: Day 2: SEC column
25. Collect peak fractions and concentrate to approximately 5-7 mg/mL for cryo-EM grid preparation