Aug 29, 2024
Purification of mCherry-ATG101/13 (1-191aa) subcomplex
- 1Sascha Martens lab, University of Vienna, Max Perutz Labs - Vienna
Protocol Citation: Elias Adriaenssens 2024. Purification of mCherry-ATG101/13 (1-191aa) subcomplex. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld8wo9v5b/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: 103083
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 mCherry-ATG101/13 (1-191aa) subcomplex.
Materials
Lysis buffer:
| A | B | |
| Tris-HCl pH 7.4 | 50 mM | |
| NaCl | 200 mM | |
| Triton X-100 | 1% | |
| glycerol | 10% | |
| MgCl2 | 2 mM | |
| β-mercaptoethanol | 2mM | |
| cOmplete EDTA-free protease inhibitors (Roche) | ||
| CIP protease inhibitor (Sigma) | ||
| Benzonase |
Wash buffer I:
| A | B | |
| Tris-HCl pH 7.4 | 50 mM | |
| NaCl | 200 mM | |
| MgCl2 | 2 mM | |
| DTT | 1mM | |
| Triton X-100 | 1% | |
| glycerol | 10% |
Wash buffer II:
| A | B | |
| Tris-HCl pH 7.4 | 50 mM | |
| NaCl | 200 mM | |
| MgCl2 | 2 mM | |
| DTT | 1mM |
Wash buffer:
| A | B | |
| Tris-HCl pH 7.4 | 50 mM | |
| NaCl | 200 mM | |
| MgCl2 | 2 mM | |
| DTT | 1 mM |
SEC buffer:
| A | B | |
| Tris-HCl pH 7.4 | 50 mM | |
| NaCl | 200 mM | |
| MgCl2 | 1 mM | |
| DTT | 1 mM |
- mCherry-tagged ATG13 (1-191aa) from a pCAG backbone (available from Addgene)
- GST-TEV-ATG101 (available from Addgene)
- FreeStyle‱ 293 Expression Medium (Thermo, 12338-026)
FreeStyle™ 293 Expression MediumThermo FisherCatalog #12338026
- 13 ml of Opti-MEMR I Reduced Serum Medium (Thermo, 31985-062)
Opti-MEM™ I Reduced Serum MediumThermo FisherCatalog #31985062
- 800 µg Polyethylenimine (PEI 25K, Polysciences CatNo 23966-1) Polyethylenimine, Linear, MW 25000, Transfection Grade (PEI 25K™)Polysciences, Inc.Catalog #23966-1
- 100 mL EXCELL R 293 Serum-Free Medium (Sigma-Aldrich, 14571C- 1000ML)EX-CELL® 293 Serum-Free Medium for HEK 293 CellsMerck MilliporeSigma (Sigma-Aldrich)Catalog #14571C
- SORVAL RC6+ centrifuge with an F21S-8x50Y rotor (Thermo Scientific)
- Glutathione Sepharose 4B beads (GE Healthcare)
- 10 kDa cut-off Amicon filter (Merck Millipore)Amicon® Ultra Centrifugal Filter, 10 kDa MWCOMerck MilliporeSigma (Sigma-Aldrich)Catalog #UFC801008
- Superdex S200 Increase 10/300 GL column (Cytiva)
Purification - mCherry-ATG101/13 (1-191aa) subcomplex
Purification - mCherry-ATG101/13 (1-191aa) subcomplex
20m
20m
To purify mCherry-ATG13/101 HORMA dimer, we express mCherry-tagged ATG13 (1-191aa) from a pCAG backbone (available from Addgene) together with GST-TEV-ATG101 (available from Addgene).
Express the ATG13/101 HORMA dimer in FreeStyleTM HEK293F cells, grow 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 µg of plasmid at a molar 1:1 ratio, dilute in 13 mL of Opti-MEMR I Reduced Serum Medium (Thermo, 31985-062), and 800 µg Polyethylenimine (PEI 25K, Polysciences CatNo 23966-1), also dilute in 13 ml of Opti-MEM media.
One day post transfection, supplement the culture with 100 mL EXCELL R 293 Serum-Free Medium (Sigma-Aldrich, 14571C- 1000ML).
Another 24 h later, harvest the cells by centrifugation at 270 x g, 00:20:00 .
20m
Wash the pellet with PBS to remove medium and then flash-frozen in liquid nitrogen.
Note
Store the pellets at -80 °C .
For purification of the ATG13/101 subcomplex, resuspend the cell pellet in 25 mL lysis buffer.
Lysis buffer:
| A | B | |
| Tris-HCl pH 7.4 | 50 mM | |
| NaCl | 200 mM | |
| Triton X-100 | 1% | |
| glycerol | 10% | |
| MgCl2 | 2 mM | |
| β-mercaptoethanol | 2mM | |
| cOmplete EDTA-free protease inhibitors (Roche) | ||
| CIP protease inhibitor (Sigma) | ||
| Benzonase |
Homogenize the cells with a douncer and clear the lysates by centrifugation at 10000 x g, 4°C, 00:45:00 with a SORVAL RC6+ centrifuge with an F21S-8x50Y rotor (Thermo Scientific).
45m
Collect the supernatant and incubate with pre-equilibrated Glutathione Sepharose 4B beads (GE Healthcare) for 02:00:00 at 4 °C with gentle shaking to bind GST-TEV-ATG101/mCherry-ATG13(1-191aa).
2h
Centrifuge the samples to pellet the beads and remove the unbound lysate.
Wash the beads twice with wash buffer I followed by three washes in wash buffer II.
Wash buffer I:
| A | B | |
| Tris-HCl pH7.4 | 50 mM | |
| NaCl | 200 mM | |
| MgCl2 | 2 mM | |
| DTT | 1mM | |
| Triton X-100 | 1% | |
| glycerol | 10% |
Wash buffer II:
| A | B | |
| Tris-HCl pH 7.4 | 50 mM | |
| NaCl | 200 mM | |
| MgCl2 | 2 mM | |
| DTT | 1mM |
Incubate the beads Overnight with TEV protease in wash buffer at 4 °C , to release mCherry- or GFP-tagged ATG13/101 from the beads.
Wash buffer:
| A | B | |
| Tris-HCl pH 7.4 | 50 mM | |
| NaCl | 200 mM | |
| MgCl2 | 2 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 the supernatant fractions, filter through a 0.45 µm syringe filter, concentrate with 10 kDa cut-off Amicon filter (Merck Millipore), and load onto a pre-equilibrated Superose S6 Increase 10/300 GL column (Cytiva).
Elute the proteins with SEC buffer.
SEC buffer:
| A | B | |
| Tris-HCl 7.4 | 50 mM | |
| NaCl | 200 mM | |
| MgCl2 | 1 mM | |
| DTT | 1 mM |
Analyze the fractions by SDS-PAGE and Coomassie staining. Pool the fractions containing both ATG13/101.
After concentrating the purified protein, aliquote the protein and snap-frozen in liquid nitrogen.
Note
Store the pellets at -80 °C .