Sep 23, 2023
Purification of MBP-NAP1
- 1Sascha Martens lab, University of Vienna, Max Perutz Labs - Vienna
Protocol Citation: Elias Adriaenssens 2023. Purification of MBP-NAP1. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1q2ykgr2/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: May 31, 2024
Protocol Integer ID: 88169
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 describes purification of MBP-TEV-NAP1 protein.
Attachments
839-2173.pdf
48KB
Materials
Reagents
- NAP1 WT (RRID:Addgene_208871)
- NAP1 delta-NDP52 (S37K/A44E) (RRID:Addgene_208872)
- NAP1 delta-TBK1 (L226Q/L233Q) (RRID:Addgene_208873)
- pGEX-4T1 vector
- 2xTY medium
- D-maltose (Santa Cruz)
- IPTG
- Amylose beads (Biolabs)
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)
- Amicon filter (Merck Millipore)
- Superose 6 Increase 10/300 GL column (Cytiva)
Purification of MBP-NAP1
Purification of MBP-NAP1
20h 46m
20h 46m
To purify MBP-NAP1, gene-synthesize human NAP1 cDNA (by Genscript) and subclone into a pGEX-4T1 vector with an N-terminal MBP-tag. Follow it by a TEV cleavage site before wild-type NAP1 (RRID:Addgene_208871), NAP1 delta-NDP52 (S37K/A44E) (RRID:Addgene_208872), or NAP1 delta-TBK1 (L226Q/L233Q) (RRID:Addgene_208873).
For expression of MBP-TEV-NAP1 in E. coli, transfer the pGEX-4T1 vector encoding MBP-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 them 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 and then clear by centrifugation.
Sonicate cell lysates for 00:00:30 . (1/2)
30s
Sonicate cell lysates for 00:00:30 . (2/2)
30s
Then, 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 Amylose beads (Biolabs) for 02:00:00 at 4 °C with gentle shaking to bind MBP-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 the beads Overnight at 4 °C with 250 millimolar (mM) D-maltose (Santa Cruz) dissolved in wash buffer.
2h
After the proteins are released from the beads, filter the MBP-TEV-NAP1 protein through a 0.45 µm syringe filter, concentrate using a 30 kDa cut-off Amicon filter (Merck Millipore), and load onto a pre-equilibrated Superose 6 Increase 10/300 GL column (Cytiva).
Elute the proteins with SEC buffer.
SEC buffer
| A | B | |
| Tris-HCl pH 7.4 | 25 mM | |
| NaCl | 300 mM | |
| DTT | 1 mM |
Analyze the fractions by SDS-PAGE and Coomassie staining.
Pool the fractions containing purified MBP-TEV-NAP1 protein.
After concentrating the purified protein, aliquot the protein and snap-freeze in liquid nitrogen. Store the proteins at -80 °C .