Sep 23, 2023
Purification of NAP1 or GST-NAP1
- 1Sascha Martens lab, University of Vienna, Max Perutz Labs - Vienna
Protocol Citation: Elias Adriaenssens 2023. Purification of NAP1 or GST-NAP1. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3xk41g25/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: September 14, 2023
Last Modified: June 01, 2024
Protocol Integer ID: 88168
Keywords: ASAPCRN, purification of gst, nap1 this protocol, unlabelled nap1, purification, nap1, gst, protocol, purification of nap1
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-000350
Marie Skłodowska-Curie MSCA Postdoctoral fellowship
Grant ID: 101062916
Abstract
This protocol describes purification of GST-NAP1 and unlabelled NAP1.
Attachments
840-2174.pdf
49KB
Materials
Materials
- pGEX-4T1 vector
- Glutathione Sepharose 4B beads (GE Healthcare)
- Superose 6 Increase 10/300 GL column (Cytiva)
- Amicon filter (Merck Millipore)
Lysis buffer
| A | B | |
| Tris-HCl pH 7.4 | 50 mM | |
| NaCl | 300 mM | |
| DTT | 1 mM | |
| MgCl2 | 2 mM | |
| glycerol | 5% | |
| β-mercaptoethanol | 2 mM | |
| cOmplete EDTA-free protease inhibitors (Roche) | ||
| DNase (Sigma) |
Wash buffer
| A | B | |
| Tris-HCl pH 7.4 | 50 mM | |
| NaCl | 300 mM | |
| glycerol | 5% | |
| DTT | 1 mM |
High-salt wash buffer
| A | B | |
| Tris-HCl pH 7.4 | 50 mM | |
| NaCl | 700 mM | |
| glycerol | 5% | |
| DTT | 1 mM |
SEC buffer
| A | B | |
| Tris-HCl pH 7.4 | 25 mM | |
| NaCl | 300 mM | |
| DTT | 1 mM |
Equipment
SORVAL RC6+ centrifuge
F21S-8x50Y rotor (Thermo Scientific)
Purification of NAP1 or GST-NAP1
18h 46m
To purify NAP1 or GST-NAP1, synthesize or clone human NAP1 cDNA in a pGEX-4T1 vector with an N-terminal GST tag followed by a TEV cleavage site (RRID:Addgene_208870).
For expression of GST-TEV-NAP1 in E. coli, transform the pGEX-4T1 vector encoding GST-TEV-NAP1 into E. coli Rosetta pLySS cells. Grow the cells in 2xTY medium at 37 °C until an OD600 of 0.4 and then continue at 18 °C .
Once the cells reached an OD600 of 0.8, induce protein expression with 50 micromolar (µM) IPTG for 16:00:00 at 18 °C .
16h
Collect the cells by centrifugation and resuspend it in lysis buffer.
Lysis buffer
| A | B | |
| Tris-HCl pH 7.4 | 50 mM | |
| NaCl | 300 mM | |
| DTT | 1 mM | |
| MgCl2 | 2 mM | |
| glycerol | 5% | |
| β-mercaptoethanol | 2 mM | |
| cOmplete EDTA-free protease inhibitors (Roche) | ||
| DNase (Sigma) |
Sonicate cell lysates.
Sonicate cell lysates for 00:00:30 . (1/2)
30s
Sonicate cell lysates for 00:00:30 . (1/2)
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 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-NAP1.
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 | |
| glycerol | 5% | |
| DTT | 1 mM |
High-salt wash buffer
| A | B | |
| Tris-HCl pH 7.4 | 50 mM | |
| NaCl | 700 mM | |
| glycerol | 5% | |
| DTT | 1 mM |
Incubate beads Overnight at 4 °C with TEV protease (for unlabeled NAP1) or 4 mL of 50 millimolar (mM) reduced glutathione dissolved in wash buffer (for GST-NAP1).
2h
After the proteins were released from the beads, filter the GST-NAP1 protein through a 0.45 µm syringe filter, concentrated using a 30 kDa cut-off Amicon filter (Merck Millipore), or 10 kDa cut-off in case of unlabeled NAP1, and loaded onto a pre-equilibrated Superose 6 Increase 10/300 GL column (Cytiva).
Elute proteins with SEC buffer.
SEC buffer
| A | B | |
| Tris-HCl pH 7.4 | 25 mM | |
| NaCl | 300 mM | |
| DTT | 1 mM |
Analyze fractions by SDS-PAGE and Coomassie staining.
Pool fractions containing purified NAP1 or GST-NAP1 protein.
After concentrating the purified protein, aliquot the protein and snap-freeze in liquid nitrogen. Store proteins at -80 °C .