Aug 29, 2024
Purification of FKBP8-GST
- 1Sascha Martens lab, University of Vienna, Max Perutz Labs - Vienna
Protocol Citation: Elias Adriaenssens 2024. Purification of FKBP8-GST. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvjne3pgk5/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: August 21, 2024
Last Modified: August 29, 2024
Protocol Integer ID: 106139
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 the purification and analysis of FKBP8-GST by SDS-PAGE and Coomassie staining.
Materials
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 | 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 |
Salt wash 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 | 300 mM | |
| DTT | 1 mM |
- Rosetta™(DE3)pLysS Competent Cells - NovagenMerckCatalog #70956-4
- pET-Duet encoding FKBP8 (1-391aa)-thrombin-GST (available from Addgene)
Purification procedure
Purification procedure
1d 2h 45m 30s
1d 2h 45m 30s
To purify FKBP8-GST, fuse the cytosol-exposed domain of FKBP8 (1-391aa) to a C-terminal GST-tag and clone into a pET-DUET1 vector.
After the transformation of the pET-DUET1 vector encoding FKBP8-GST 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 reach 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 by 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 | 2 mM | |
| cOmplete EDTA-free protease inhibitors (Roche) | ||
| CIP protease inhibitor (Sigma) | ||
| DNase (Sigma) | ||
Sonicate the cell lysates twice for 00:00:30 and clear by centrifugation at 18000 rpm, 4°C, 00:45:00 in a SORVAL RC6+ centrifuge with an F21S-8x50Y rotor (Thermo Scientific).
- 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 FKBP8-GST.
2h 45m 30s
Centrifuge the samples to pellet the beads and remove the unbound lysate. Then 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 |
Salt wash Buffer:
| A | B | |
| Tris-HCl pH 7.4 | 50 mM | |
| NaCl | 700 mM | |
| DTT | 1 mM |
Incubate the beads Overnight with 4 mL of 50 millimolar (mM) reduced glutathione dissolved in wash buffer at 4 °C , to elute FKBP8-GST from the beads.
Wash Buffer:
| A | B | |
| Tris-HCl pH 7.4 | 50 mM | |
| NaCl | 300 mM | |
| DTT | 1 mM |
8h
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 30 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.
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 FKBP8-GST.
After concentrating the purified protein, aliquot the protein and snap-frozen in liquid nitrogen.
Store proteins at -80 °C .