Jul 20, 2022
Purification of human ATP13A2 for cryo-EM analysis
- 1University of California, Berkeley
Protocol Citation: Sue Sim, eunyong_park 2022. Purification of human ATP13A2 for cryo-EM analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.261genmojg47/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: July 17, 2022
Last Modified: July 20, 2022
Protocol Integer ID: 66878
Abstract
Purification of GFP-tagged human ATP13A2 expressed in Sf9 cells for cryo-EM analysis
Materials
Lysis Buffer
50 mM Tris pH 7.5
200 mM NaCl
1 mM EDTA
1 mM DTT
10% glycerol
Plus protease inhibitors (5 µg/mL aprotinin, 5 µg/mL leupeptin, 1 µg/mL pepstatin A, and 2 mM PMSF)
Wash Buffer
25 mM Tris pH 7.5
100 mM NaCl
1 mM EDTA
1 mM DTT
0.03% DDM/ 0.006% CHS
Day 1: Crude membrane preparation and solubilization
Day 1: Crude membrane preparation and solubilization
7h
7h
Thaw Sf9 cell pellets at room temperature (typical size around 10 g from 0.7L of culture)
All subsequent steps performed at 4 °C
Resuspend each pellet in 30 mL Lysis Buffer (use 3x volume of cell pellet, 40 mL total volume)
Homogenize pellet with Dounce homogenizer, 100 plunges tight on ice
Pour lysate into pre-chilled 50 mL centrifuge tubes
Spin in centrifuge at 4000 x g, 4°C, 00:10:00 to remove unbroken cells
10m
Transfer supernatant to pre-chilled ultracentrifuge rotor tubes
Spin lysate in ultracentrifuge at 100000 x g, 4°C, 01:30:00 to pellet membranes (Beckman Type 45 Ti rotor)
1h 30m
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)
Solubilize by rotating end-over-end for 02:30:00 at 4 °C
2h 30m
Clarify lysate in ultracentrifuge at 100000 x g, 4°C, 01:00:00
1h
Day 1: Bead binding and overnight 3C cleavage
Day 1: Bead binding and overnight 3C cleavage
3h
3h
Equilibrate 1 mL Sepharose beads conjugated with anti-GFP nanobody with Wash Buffer
Add beads to gravity column and wash with 10 mL wash buffer
After ultracentrifugation is complete, transfer supernatant into 50 mL falcon tube
Add 1 mL equilibrated anti-GFP nanobody beads to tube
Incubate by rotating end-over-end for 02:30:00 at 4 °C
2h 30m
Transfer to gravity column and let flow-through drain
Wash beads with 30 column volumes of Wash Buffer
Add 5 mL Wash Buffer to gravity column and 10 µg/mL HRV 3C protease
Incubate by rotating end-over-end overnight at 4 °C
Day 2: SEC column
Day 2: SEC column
3h
3h
Equilibrate Superose 6 Increase 10/300 GL column with Wash Buffer
Concentrate the protein to 0.5 mL using an Amicon Ultrafilter (cut-off 100kDa)
After concentration, spin protein at 17000 x g, 4°C, 00:10:00
10m
Injected sample into FPLC
Collect peak fractions and concentrate to approximately 5-7 mg/mL for cryo-EM grid preparation