Aug 29, 2024
Purification mCherry-ATG13 IDR
- 1Sascha Martens lab, University of Vienna, Max Perutz Labs - Vienna
Protocol Citation: Elias Adriaenssens 2024. Purification mCherry-ATG13 IDR. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbz4m1gpk/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 02, 2024
Last Modified: August 29, 2024
Protocol Integer ID: 103081
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-ATG13 IDR.
Materials
Lysis buffer:
| A | B | |
| Tris-HCl pH 7.4 | 50 mM | |
| pH | 7.4 | |
| NaCl | 300 mM | |
| MgCl2 | 2 mM | |
| glycerol | 5% | |
| Triton X-100 | 1% | |
| Imidazole | 10 mM | |
| β-mercaptoethanol | 2 mM | |
| cOmplete EDTA-free protease inhibitors (Roche) | ||
| CIP protease inhibitor (Sigma) | ||
| DNase (Sigma) | ||
Wash buffer:
| A | B | |
| Tris-HCl pH 7.4 | 50 mM | |
| NaCl | 300 mM | |
| Imidazole | 10 mM | |
| β-mercaptoethanol | 2 mM |
SEC Buffer:
| A | B | |
| Tris-HCl pH 7.4 | 25 mM | |
| NaCl | 150 mM | |
| DTT | 1 mM |
Purification
Purification
16h
16h
To purify mCherry-tagged ATG13 IDR, fuse the coding sequence for ATG13 (190-517aa) or ATG13 (230-517aa) to a N-terminal 6xHis-TEV-mCherry-tag through cloning into a pET-DUET1 vector (plasmids available from Addgene).
After the transformation of the pET-DUET1 vectors encoding the mCherry-tagged ATG13 IDR in E. coli Rosetta pLysS cells (Novagen Cat# 70956-4), grow the cells in 2x Tryptone Yeast extract (TY) medium at 37 °C until an OD600 of 0.4 and then continue at 18 °C .
Once the cells reaches an OD600 of 0.8, induce the protein expression with 100 micromolar (µM) isopropyl β-D-1-thiogalactopyranoside (IPTG) for 16:00:00 at 18 °C .
16h
Collect cells by centrifugation and resuspend in lysis buffer.
Lysis buffer:
| A | B | |
| Tris-HCl pH 7.4 | 50 mM | |
| pH | 7.4 | |
| NaCl | 300 mM | |
| MgCl2 | 2 mM | |
| glycerol | 5% | |
| Triton X-100 | 1% | |
| Imidazole | 10 mM | |
| β-mercaptoethanol | 2 mM | |
| cOmplete EDTA-free protease inhibitors (Roche) | ||
| CIP protease inhibitor (Sigma) | ||
| DNase (Sigma) | ||
Sonicate the cell lysates twice for 00:00:30 .
30s
Clear the lysates by centrifugation at 18000 rpm, 4°C, 00:45:00 in a SORVAL RC6+ centrifuge with an F21S-8x50Y rotor (Thermo Scientific).
45m
Filter the supernatant through an 0.45 µm filter and load onto a pre-equilibrated 5 mL His-Trap HP column (Cytiva).
After His-tagged proteins are bound to the column, wash the column with three column volumes of wash buffer.
Wash buffer:
| A | B | |
| Tris-HCl pH 7.4 | 50 mM | |
| NaCl | 300 mM | |
| Imidazole | 10 mM | |
| β-mercaptoethanol | 2 mM |
Elute the proteins with a stepwise imidazole gradient (30, 75, 100, 150, 225, 300 mM).
Pool the fractions containing the 6xHis-TEV-mCherry-ATG13 IDR, concentrated using a 30 kDa cut-off Amicon filter (Merck Millipore).
Load the samples were ed onto a pre-equilibrated Superose 200 Increase 10/300 GL column (Cytiva). Elute the proteins with SEC buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM DTT).
SEC Buffer:
| A | B | |
| Tris-HCl pH 7.4 | 25 mM | |
| NaCl | 150 mM | |
| DTT | 1 mM |
Analyze the fractions by SDS-PAGE and Coomassie staining.
Pool the fractions containing purified ATG13 IDR.
After concentrating the purified protein, aliquote the protein and snap-frozen in liquid nitrogen.
Store the proteins at -80 °C .