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 @ 4°C end-over-end for 30 minutes.
5. Centrifuge at 12,000g for 10 min @ 4°C.
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 @ 4°C 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 @ 4°C.
b) If eluting from beads, add 1 volume GST elution buffer, incubate end-overend for 10 min @ 4°C. 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 μM CaCl2 (or MgCl2) and 0.1% βME
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 μl protease in 480 μl buffer]
3. Incubate end-over-end for 4 hours (usually overnight) @ 4°C.
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 -80°C.
PreScission™ Protease Cleavage Buffer:
Stock
Solution
Concentration
1M
5M
500mM
1M
10%
Volume of
Stock
Final
Concentration
Tris pH 8.0
NaCl
EDTA
DTT
NP-40
2.5mL
50mM
1.5mL
150mM
100uL
1mM
50uL
1mM
50uL
0.01%
MQ dH2O
to 50mL