Aug 29, 2024
Purification GFP-ATG13 IDR
- 1Sascha Martens lab, University of Vienna, Max Perutz Labs - Vienna
Protocol Citation: Elias Adriaenssens 2024. Purification GFP-ATG13 IDR. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5rey4g1b/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: 103077
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 GFP-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% | |
| 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 | |
| DTT | 1 mM |
High salt 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
Purification
16h
16h
To purify GFP-tagged ATG13 IDR, the coding sequence for ATG13 (190-517aa), (206-517aa), (231-517aa), (190-205_231-517aa), (190-230aa), (190-205aa), or (206-230aa) into GST-TEV-EGFP-insert through cloning into a pGEX-4T1 vector (Plasmids available from Addgene).
Mutants
- 3A (M196A/S197A/R199A) and
- 11A (M196A/S197A/R199A/G202A/T204A/P205A/I207A/M208A/I210A/D213A/H214A)
are also expressed according to the protocol below.
After the transformation of the pGEX-4T1 vectors encoding the GFP-tagged ATG13 IDR in E. coli Rosetta pLysS cells (Novagen Cat# 70956-4), grow 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-t isopropyl β-D-1-thiogalactopyranoside (IPTG) for 16:00:00 at 18 °C .
16h
Collect cells by centrifugation and resuspend in lysis buffer for GST-tagged proteins (50 mM Tris-HCl pH 7.4, 300 mM NaCl, 2 mM MgCl2, 5% glycerol, 1% Triton X-100, 2 mM β-mercaptoethanol, cOmplete EDTA-free protease inhibitors (Roche), CIP protease inhibitor (Sigma), and DNase (Sigma)).
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% | |
| 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
Collect the supernatant after centrifugation and incubate with pre-equilibrated Glutathione Sepharose 4B beads (GE Healthcare) for 02:00:00 at 4 °C on a roller to bind GST-TEV-EGFP-ATG13 IDR.
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 |
High salt buffer:
| A | B | |
| Tris-HCl pH 7.4 | 50 mM | |
| NaCl | 700 mM | |
| DTT | 1 mM |
Incubate the beads Overnight with TEV protease at 4 °C , to elute GFP-tagged ATG13 IDR from the beads.
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, concentrated with 10 or 30 kDa cut-off Amicon filter (Merck Millipore).
Load the samples onto a pre-equilibrated Superose 200 Increase 10/300 GL column (Cytiva) or S75 Increase 10/300 column (Cytiva) in case of the smaller peptides (190-230aa and smaller variants thereof).
Elute the proteins with SEC buffer.
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 was and snap-frozen in liquid nitrogen.
Store the proteins at -80 °C .