Raught Lab 2008 GST-tagged Protein Purification: 1.

Resuspend pellet in 50uL of ice cold 1xPBS per ml pellet (for 1L overnight liquid culture, pellet is approximately 5ml, but 40mL of PBS is used to resuspend the pellet). 2. Disrupt using sonicator (4-6x 30sec bursts, with ~1min rest on ice in-between) AVOID frothing. 3. Transfer the lysate to 40mL centrifuge tube. 4. Add Triton X-100 to 2% (800uL for 40mL), ensure that the Triton X-100 is dissolved then incubate @ 4C end-over-end for 30 minutes. 5. Centrifuge at 12,000g for 10 min @ 4C. 6. While centrifuging wash beads with PBS 3 times (2mL MagneGST beads for each 100mL of sonicate) {we normally use 520uL of MagneGST beads for 1L of culture} 7. Transfer the centrifuged lysate to the tube containing the pre-washed beads. 8. Incubate end-over-end @ 4C for 2-3 hours. 9. Wash beads with PBS 3 times. 10. a) If storing beads, store in 1 bead volume of PBS supplemented with 5% glycerol @ 4C. b) If eluting from beads, add 1 volume GST elution buffer, incubate end-overend for 10 min @ 4C. Collect supernatant. Repeat once more with the GST elution buffer and pool with previous supernatant. Repeat one last time with the supplemented GST elution buffer and pool with previous supernatants. c) If cleaving free of GST tag, see PreScission Cleavage protocol.

GST Elution Buffer Recipe: 42 mg reduced dry glutathione in 40 ml of 50 mM TrisCL pH 8.0 Filter through atleast 45um syringe filter and aliquot and store 6 months at -20 C Supplemented GST elution buffer: Add of 150 mM NaCl, 5 M CaCl2 (or MgCl2) and 0.1% ME

Raught Lab 2008 PreScission Protease Cleavage: 1. Wash beads 3x with 2 beads volumes of PreScission Protease cleavage buffer (1mL normally) 2. Add 1 bead volume of cleavage solution [4% protease stock (2,000 units/ml) in cleavage buffer i.e. 20 l protease in 480 l buffer] 3. Incubate end-over-end for 4 hours (usually overnight) @ 4C. 4. Collect the supernatant. 5. Add 1 bead volume of cleavage buffer, and incubate end-over-end for 10 min. 6. Pool supernatant together. Store at -80C.

PreScission Protease Cleavage Buffer:

Stock Solution Concentration 1M 5M 500mM 1M 10% Volume of Stock Final Concentration 50mM 150mM 1mM 1mM 0.01%

Tris pH 8.0 NaCl EDTA DTT NP-40 MQ dH2O

2.5mL 1.5mL 100uL 50uL 50uL to 50mL