Feb 01, 2024
Purification of GST-LC3B
- 1Sascha Martens lab, University of Vienna, Max Perutz Labs - Vienna
Protocol Citation: Elias Adriaenssens 2024. Purification of GST-LC3B. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4qnbjvo5/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
Created: January 31, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 94565
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 of GST-LC3B.
Attachments
ppgyb32rp.pdf
29KB
Materials
Materials:
- pGEX-4T1 vector (LC3B-encoding vector is available from Addgene)
- Glutathione Sepharose 4B beads (GE Healthcare)
- Amicon filter (Merck Millipore)
- Superdex 75 16/600 column (Cytiva)
Lysis buffer:
| A | B | |
| HEPES pH 7.5 | 50 mM | |
| NaCl | 300 mM | |
| MgCl2 | 2 mM | |
| βmercaptoethanol | 2 mM | |
| cOmplete EDTA-free protease inhibitors (Roche) | ||
| DNase (Sigma) |
Wash buffer:
| A | B | |
| HEPES pH 7.4 | 50 mM | |
| NaCl | 300 mM | |
| DTT | 1 mM |
High-salt wash buffer:
| A | B | |
| HEPES pH 7.4 | 50 mM | |
| NaCl | 700 mM | |
| DTT | 1 mM |
SEC buffer:
| A | B | |
| HEPES pH 7.4 | 25 mM | |
| NaCl | 150 mM | |
| DTT | 1 mM |
Equipment
- Beckman Ti45 rotor
Purification of GST-LC3B
Purification of GST-LC3B
8h 30m 30s
To purify GST-LC3B, insert human LC3B cDNA in a pGEX-4T1 vector. Note, the last five amino acids of LC3B are deleted to mimic the cleavage by ATG4.
After the transformation of the pGEX-4T1 vector encoding GST-LC3B in E.coli Rosetta (DE3) pLysS cells, grow the cells in LB medium at 37 °C until an OD600 of 0.8-1 and induce the protein expression with 1 millimolar (mM) IPTG for 04:00:00 at 37 °C .
4h
Collect cells by centrifugation and resuspend in lysis buffer.
Lysis buffer:
| A | B | |
| HEPES pH 7.5 | 50 mM | |
| NaCl | 300 mM | |
| MgCl2 | 2 mM | |
| βmercaptoethanol | 2 mM | |
| cOmplete EDTA-free protease inhibitors (Roche) | ||
| DNase (Sigma) |
Sonicate cell lysates.
1m
Sonicate cell lysates twice for 00:00:30 (1/2).
30s
Sonicate cell lysates twice for 00:00:30 (2/2).
30s
Clear lysates by centrifugation at 140000 x g, 4°C, 00:30:00 in a Beckman Ti45 rotor.
30m
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-LC3B.
2h
Centrifuge the samples to pellet the beads and remove the unbound lysate.
Wash beads twice with wash buffer, once with high salt wash buffer and two more times with wash buffer.
Wash buffer
| A | B | |
| HEPES pH 7.4 | 50 mM | |
| NaCl | 300 mM | |
| DTT | 1 mM |
High-salt wash buffer
| A | B | |
| HEPES pH 7.4 | 50 mM | |
| NaCl | 700 mM | |
| DTT | 1 mM |
Elute the proteins Overnight with 20 millimolar (mM) reduced L-glutathione in 50 millimolar (mM) HEPES 7.4 , 300 millimolar (mM) NaCl, 1 millimolar (mM) DTT buffer.
8h
Collect the supernatant, filter through a 0.45 µm syringe filter, concentrate using a 10 kDa cut-off Amicon filter (Merck Millipore), and load onto a pre-equilibrated Superdex 75 16/600 column (Cytiva).
Elute the proteins with SEC buffer.
SEC buffer
| A | B | |
| HEPES pH 7.4 | 25 mM | |
| NaCl | 150 mM | |
| DTT | 1 mM |
Analyze fractions by SDS-PAGE and Coomassie staining.
Pool fractions containing purified GST-LC3B protein.
After concentrating the purified protein, aliquot the protein and snap-frozen it in liquid nitrogen. Store proteins at -80 °C .