Sep 23, 2023
Purification of GST-tagged linear tetra-ubiquitin (4xUb)
- 1Sascha Martens lab, University of Vienna, Max Perutz Labs - Vienna
Protocol Citation: Elias Adriaenssens 2023. Purification of GST-tagged linear tetra-ubiquitin (4xUb). protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7pbo1gwz/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 07, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 84631
Keywords: GST-tagged linear tetra-ubiquitin, purification, 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 GST-tagged linear tetra-ubiquitin (4xUb).
Attachments
774-1960.pdf
59KB
Materials
Materials
- pGEX-4T1 vector (RRID:Addgene #199779)
- isopropyl β-D-1-thiogalactopyranoside (IPTG)
- Glutathione Sepharose 4B beads (GE Healthcare)
- 10 kDa cut-off Amicon filter (Merck Millipore)
- Superdex 200 Increase 10/300 GL column (Cytiva)
- SORVAL RC6+ centrifuge with an F21S8x50Y rotor (Thermo Scientific)
Lysis buffer
| A | B | |
| Tris-HCl pH 7.4 | 50 mM | |
| NaCl | 300 mM | |
| MgCl2 | 2 mM | |
| DTT | 1 mM | |
| cOmplete EDTA-free protease inhibitors (Roche) | ||
| DNase (Sigma) | ||
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 |
SEC buffer
| A | B | |
| Tris-HCl pH 7.4 | 25 mM | |
| NaCl | 150 mM | |
| DTT | 1 mM |
Purification of GST-tagged linear tetra-ubiquitin (4xUb)
Purification of GST-tagged linear tetra-ubiquitin (4xUb)
18h 46m
18h 46m
Linear tetra-ubiquitin fused to GST (GST-4xUb) was cloned into a pGEX-4T1 vector and is available from Addgene (RRID:Addgene #199779).
After the transformation of the pGEX-4T1 vector encoding GST4xUb in E. coli Rosetta pLySS cells, grow cells in LB medium at 37 °C until an OD600 of 0.4 and then continued at 18 °C .
Once the cells reached an OD600 of 0.8, induce protein expression with 100 micromolar (µM) isopropyl β-D-1-thiogalactopyranoside (IPTG) for 16:00:00 at 18 °C .
16h
Collect cells by centrifugation and resuspend in lysis buffer.
Sonicate cell lysates.
Sonicate cell lysates for 00:00:30 . (1/2)
30s
Sonicate cell lysates for 00:00:30 . (2/2)
30s
Clear lysates by centrifugation at 18000 rpm, 4°C, 00:45:00 in a SORVAL RC6+ centrifuge with an F21S8x50Y rotor (Thermo Scientific).
45m
Collect the supernatant and incubate with preequilibrated Glutathione Sepharose 4B beads (GE Healthcare) for 02:00:00 at 4 °C with gentle shaking to bind GST-4xUb.
2h
Centrifuge the samples to pellet the beads and remove the unbound lysate.
Wash the beads.
Wash the beads twice with wash buffer.
Wash the beads once with high salt wash buffer.
Wash the beads twice with wash buffer.
Incubate the beads Overnight with 4 mL of 50 millimolar (mM) reduced glutathione dissolved in wash buffer at 4 °C , to elute GST-4xUb from the beads.
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 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.
Pool the fractions containing purified GST-4xUb.
After concentrating the purified protein, aliquot the protein and snap-freeze in liquid nitrogen. Store the proteins at -80 °C .