Aug 29, 2024
Purification of NIX-GFP
- 1Sascha Martens lab, University of Vienna, Max Perutz Labs - Vienna
Protocol Citation: Elias Adriaenssens 2024. Purification of NIX-GFP. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9219zl3e/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: 103094
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 NIX-GFP.
Materials
- Lysis buffer:
| A | B | |
| Tris-HCl | 50 mM | |
| pH | 7.4 | |
| NaCl | 300 mM | |
| Triton X-100 | 1% | |
| glycerol | 5% | |
| MgCl2 | 2 mM | |
| DTT | 1 mM | |
| β-mercaptoethanol | 2mM | |
| cOmplete EDTA-free protease inhibitors (Roche) | ||
| CIP protease inhibitor (Sigma) | ||
| DNase (Sigma) |
Wash buffer:
| Tris-HCl | 50 mM | |
| pH | 7.4 | |
| NaCl | 300 mM | |
| DTT | 1 mM |
High salt wash buffer:
| Tris-HCl | 50 mM | |
| pH | 7.4 | |
| NaCl | 700 mM | |
| DTT | 1 mM |
SEC buffer:
| Tris-HCl | 25 mM | |
| pH | 7.4 | |
| NaCl | 300 mM | |
| DTT | 1 mM |
Materials:
- pET-DUET1 vector (available from Addgene). pETDuet-1 TIM9,10addgeneCatalog #170280
- NIX E72A/L75A/D77A/E81A (4A; ΔWIPI2) (available from Addgene)
- NIX W35A/L38A (ΔLIR) (available from Addgene).
- Rosetta pLysS cells (Novagen Cat# 70956-4)Rosetta™(DE3)pLysS Competent Cells - NovagenMerckCatalog #70956-4
- 10 kDa cut-off Amicon filter (Merck Millipore)Amicon® Ultra Centrifugal Filter, 10 kDa MWCOMerck MilliporeSigma (Sigma-Aldrich)Catalog #UFC801008
Purification - NIX-GFP
Purification - NIX-GFP
20h 46m
20h 46m
To purify GFP-tagged
- NIX-GFP (available from Addgene) or NIX(W36A/L39A)-GFP (ΔLIR)(available from Addgene),
fuse the cytosol-exposed domain of NIX (1-182aa) to a C-terminal GFP-tag through cloning into a pET-DUET1 vector (available from Addgene).
Introduce the point mutants in vitro mutagenesis to generate
- NIX E72A/L75A/D77A/E81A (4A; ΔWIPI2) (available from Addgene), and
- NIX W36A/L39A (ΔLIR) (available from Addgene).
After the transformation of the pET-DUET1 vector encoding NIX-GFP wild-type or mutants 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 the cells centrifugation and resuspend in lysis buffer.
Lysis buffer:
| A | B | |
| Tris-HCl pH 7.4 | 50 mM | |
| NaCl | 300 mM | |
| Triton X-100 | 1% | |
| Glycerol | 5% | |
| MgCl2 | 2 mM | |
| DTT | 1 mM | |
| β-mercaptoethanol | 2mM | |
| cOmplete EDTA-free protease inhibitors (Roche) | ||
| CIP protease inhibitor (Sigma) | ||
| DNase (Sigma) |
Sonicate the cell lysates twice for 30 s and clears by centrifugation at 18.000 rpm, 4°C, 00:45:00 in a SORVAL RC6+ centrifuge with an F21S-8x50Y rotor (Thermo Scientific).
45m
Sonicate the cell lysates for 00:00:30 (1/2).
30s
Sonicate the cell lysates for 00:00:30 (2/2).
30s
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 NIX-GFP.
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 wash buffer:
| A | B | |
| Tris-HCl pH 7.4 | 50 mM | |
| NaCl | 700 mM | |
| DTT | 1 mM |
Cleave off the GST-tag Overnight by eluting the GFP-tagged cargo receptor from the GSH beads by the addition of TEV protease in wash buffer at 4 °C .
Wash buffer:
| A | B | |
| Tris-HCl pH 7.4 | 50 mM | |
| NaCl | 300 mM | |
| DTT | 1 mM |
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 Superdex 200 Increase 10/300 GL column (Cytiva). Elute the proteins with SEC buffer. Analyze the fractions by SDS-PAGE and Coomassie staining. Pools fractions containing purified NIX-GFP.
SEC buffer:
| A | B | |
| Tris-HCl pH 7.4 | 25 mM | |
| NaCl | 300 mM | |
| DTT | 1 mM |
After concentrating the purified protein, aliquote the protein and snap-frozen in liquid nitrogen.
Note
Store the proteins at -80 °C .