Aug 29, 2024
Purification of GST-WIPI1/WIPI2d/WIPI3/WIPI4
- 1Sascha Martens lab, University of Vienna, Max Perutz Labs - Vienna
Protocol Citation: Elias Adriaenssens 2024. Purification of GST-WIPI1/WIPI2d/WIPI3/WIPI4. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvjnnqxgk5/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: May 24, 2024
Last Modified: August 29, 2024
Protocol Integer ID: 101115
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-000350
Marie Skłodowska-Curie MSCA Postdoctoral fellowship
Grant ID: 101062916
Abstract
This protocol details the purification of GST-tagged WIPI1/2d/3/4.
Materials
FreeStyle™ 293 Expression MediumThermo Fisher ScientificCatalog #12338026
Opti-MEM™ I Reduced Serum MediumThermo Fisher ScientificCatalog #31985062
Polyethylenimine, Linear, MW 25000, Transfection Grade (PEI 25K™)Polysciences, Inc.Catalog #23966-1
EX-CELL® 293 Serum-Free Medium for HEK 293 CellsMerck MilliporeSigma (Sigma-Aldrich)Catalog #14571C
25ml lysis buffer:
| A | B | |
| Tris-HCl, pH 7.4 | 50 mM | |
| NaCl | 300 mM | |
| MgCl2 | 2 mM | |
| Glycerol | 5% | |
| Triton X-100 | 1% | |
| β-mercaptoethanol | 2 mM |
Wash buffer:
| A | B | |
| Tris-HCl, pH 7.4 | 50 mM | |
| NaCl | 300 mM | |
| DTT | 1 mM |
Salt wash buffer:
| A | B | |
| Tris-HCl, pH 7.4 | 50 mM | |
| NaCl | 700 mM | |
| DTT | 1 mM |
SEC buffer:
| A | B | |
| Tris-HCl, pH 7.4 | 25 mM | |
| NaCl | 150 mM | |
| DTT | 1 mM |
Purification procedure
Purification procedure
1d 5h 5m 30s
1d 5h 5m 30s
To purify GST-WIPI1/GST-WIPI2/GST-WIPI3/GST-WIPI4, we express the GST-tagged WIPI1/2d/3/4 from a pCAG backbone encoding GST-TEV-WIPI1/2/3/4 (available from Addgene).
Express the protein in FreeStyleTM HEK293F cells, grown at 37 °C in FreeStyle™ 293 Expression Medium (Thermo, 12338-026).
The day before transfection, seed the cells at a density of 0.7 x 10∧6 cells per ml.
On the day of transfection, transfect a 400 mL culture with 400 undetermined of the MAXI-prep DNA, diluted in 13 mL of Opti-MEMR I Reduced Serum Medium (Thermo, 31985-062), and 800 undetermined Polyethylenimine (PEI 25K, Polysciences CatNo 23966-1), also diluted in 13 mL of Opti-MEM media.
One day post transfection, supplement the culture with100 mL EXCELL R 293 Serum-Free Medium (Sigma-Aldrich, 14571C- 1000ML).
Another 24:00:00 later, harvest the cells by centrifugation at 270 x g, 00:20:00 .
1d 0h 20m
Wash the pellet with PBS to remove medium and then flash-frozen in liquid nitrogen.
Store the pellets at -80 °C .
For purification of GST-TEV-WIPI1/2/3/4, resuspend the cell pellet in, (complete EDTA-free protease inhibitors (Roche), CIP protease inhibitor (Sigma), and DNase (Sigma)).
25ml lysis buffer:
| A | B | |
| Tris-HCl, pH 7.4 | 50 mM | |
| NaCl | 300 mM | |
| MgCl2 | 2 mM | |
| Glycerol | 5% | |
| Triton X-100 | 1% | |
| β-mercaptoethanol | 2 mM |
Sonicate the cell lysates twice for 00:00:30 .
30s
Clear the cell lysates by centrifugation at 10000 rpm, 4°C, 00:45:00 with a SORVAL RC6+ centrifuge with an F21S-8x50Y rotor (Thermo Scientific).
45m
Collect and incubate the supernatant with pre-equilibrated Glutathione Sepharose 4B beads (GE Healthcare) for 02:00:00 at 4 °C with gentle shaking to bind GST-TEV-WIPI1/2/3/4.
2h
Centrifuge the samples to pellet the beads and remove the unbound lysate.
Wash the beads twice with wash buffer, once with high salt wash buffer, and two more times with wash buffer.
Wash buffer:
| A | B | |
| Tris-HCl, pH 7.4 | 50 mM | |
| NaCl | 300 mM | |
| DTT | 1 mM |
Salt wash buffer:
| A | B | |
| Tris-HCl, pH 7.4 | 50 mM | |
| NaCl | 700 mM | |
| DTT | 1 mM |
Incubate the beads Overnight with 4 mL of 50 millimolar (mM) reduced glutathione dissolved in wash buffer at 4 °C , to elute GST-tagged WIPI1/2/3/4 from the beads.
Wash buffer:
| A | B | |
| Tris-HCl, pH 7.4 | 50 mM | |
| NaCl | 300 mM | |
| DTT | 1 mM |
2h
To collect the supernatant, collect the beads by centrifugation.
Wash the beads twice with 4 mL of wash buffer, and collect the supernatant.
Pool and filterate the supernatant fractions through a 0.45 µm syringe filter, concentrate with 30 kDa cut-off Amicon filter (Merck Millipore), and load onto a pre-equilibrated Superdex 200 Increase 10/300 GL column (Cytiva).
Elute the proteins with SEC buffer.
SEC buffer:
| A | B | |
| Tris-HCl, pH 7.4 | 25 mM | |
| NaCl | 150 mM | |
| DTT | 1 mM |
Analyse the fractions by SDS-PAGE and Coomassie staining.
Pool the fractions containing purified GST-TEV-WIPI1/2/3/4.
After concentrating the purified protein, aliquot the protein and snap-frozen in liquid nitrogen.
Store the proteins at -80 °C .