TITLE: Purification of GST-tagged linear tetra-ubiquitin (4xUb)
AUTHORS: Elias Adriaenssens

[DESCRIPTION]
This protocol describes purification of GST-tagged linear tetra-ubiquitin (4xUb).

[STEPS]
SECTION: Purification of GST-tagged linear tetra-ubiquitin (4xUb)
1. Linear tetra-ubiquitin fused to GST (GST-4xUb) was cloned into a pGEX-4T1 vector and is available from Addgene (RRID:Addgene #199779).
SECTION: Purification of GST-tagged linear tetra-ubiquitin (4xUb)
2. 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.

SECTION: Purification of GST-tagged linear tetra-ubiquitin (4xUb)
3. Once the cells reached an OD600 of 0.8, induce protein expression with 100 micromolar (µM) isopropyl β-D-1-thiogalactopyranoside (IPTG) for 960 min at 18 °C.

SECTION: Purification of GST-tagged linear tetra-ubiquitin (4xUb)
4. Collect cells by centrifugation and resuspend in lysis buffer.
SECTION: Purification of GST-tagged linear tetra-ubiquitin (4xUb)
5. Sonicate cell lysates.
SECTION: Purification of GST-tagged linear tetra-ubiquitin (4xUb)
6. Clear lysates by centrifugation at 18000 rpm, 45 min, 4 °C in a SORVAL RC6+ centrifuge with an F21S8x50Y rotor (Thermo Scientific).
SECTION: Purification of GST-tagged linear tetra-ubiquitin (4xUb)
5.1. Sonicate cell lysates for 30 s. (1/2)

SECTION: Purification of GST-tagged linear tetra-ubiquitin (4xUb)
5.2. Sonicate cell lysates for 30 s. (2/2)
SECTION: Purification of GST-tagged linear tetra-ubiquitin (4xUb)
7. Collect the supernatant and incubate with preequilibrated Glutathione Sepharose 4B beads (GE Healthcare) for 120 min at 4 °C with gentle shaking to bind GST-4xUb.
SECTION: Purification of GST-tagged linear tetra-ubiquitin (4xUb)
8. Centrifuge the samples to pellet the beads and remove the unbound lysate.
SECTION: Purification of GST-tagged linear tetra-ubiquitin (4xUb)
9. Wash the beads. 
SECTION: Purification of GST-tagged linear tetra-ubiquitin (4xUb)
9.1. Wash the beads twice with wash buffer.
SECTION: Purification of GST-tagged linear tetra-ubiquitin (4xUb)
9.2. Wash the beads once with high salt wash buffer.
SECTION: Purification of GST-tagged linear tetra-ubiquitin (4xUb)
9.3. Wash the beads twice with wash buffer.
SECTION: Purification of GST-tagged linear tetra-ubiquitin (4xUb)
10. Incubate the beads 120 min with 4 mL of 50 millimolar (mM) reduced glutathione dissolved in wash buffer at 4 °C, to elute GST-4xUb from the beads.
SECTION: Purification of GST-tagged linear tetra-ubiquitin (4xUb)
11. To collect the supernatant, collect the beads by centrifugation.
SECTION: Purification of GST-tagged linear tetra-ubiquitin (4xUb)
12. Wash the beads twice with 4 mL of wash buffer, and collect the supernatant.

SECTION: Purification of GST-tagged linear tetra-ubiquitin (4xUb)
13. Pool the supernatant fractions, filter through a   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).

SECTION: Purification of GST-tagged linear tetra-ubiquitin (4xUb)
14. Elute the proteins with SEC buffer.
SECTION: Purification of GST-tagged linear tetra-ubiquitin (4xUb)
15. Analyze the fractions by SDS-PAGE and Coomassie staining.
SECTION: Purification of GST-tagged linear tetra-ubiquitin (4xUb)
16. Pool the fractions containing purified GST-4xUb.
SECTION: Purification of GST-tagged linear tetra-ubiquitin (4xUb)
17. After concentrating the purified protein, aliquot the protein and snap-freeze in liquid nitrogen. Store the proteins at -80 °C.