GST Purification protocol
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GST purification protocol -grow BL21 containing pGEX(insert) in 50 mL LB/amp overnight. -add 50 mL to 250 mL LB/amp, incubate 2-4 hrs. -add IPTG (to 200-400 uM final conc. Incubate 3-4 hrs. -harvest and centrifuge culture at 5,000 rpm for 7 min. (pellet can be frozen at -80C at this point). -resuspend pellet in GST buffer (10mL for every 300 mL BL21 culture) -transfer to 50 mL ultracentrifuge tube. -sonicate: 01, 01, 50% for 1 min, twice. Keep on ice. -centrifuge at 20,000 rpm for 40 min. -harvest protein supernatant into 15 mL conical vial. -add 100 uL glutathione beads. -incubate, spinning, for 2 hr at 4 ºC. -centrifuge at 2,200 rpm for 1 min. -remove supernatant (most). -wash with 5 mL GST buffer. -incubate, spinning, for 5 min. at 4 ºC. Spin down. -remove supernatant (leave ~1ml of supernatant). -using snipped tip, transfer beads to 1.5 mL tube. -centrifuge at 2,200 rpm for 30 sec. -wash with 1 mL GST buffer, spinning, 30 sec at 4 ºC. repeat the washing steps 2 more times. -centrifuge again. -remove enough supernatant to result in 50% slurry. -resuspend beads and take 10 ul for coomassie stain. -use the remaining beads for GST pulldown. GST buffer: 150 mM NaCl 20 mM Tris pH 7.4 0.5% Triton 0.1 % EDTA filter sterilize. IPTG: 0.95g in 20 ml filter sterilize with 0.2mm filter.
description
Protocol used for GST purification
Transcript of GST Purification protocol
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GST purification protocol
-grow BL21 containing pGEX(insert) in 50 mL LB/amp overnight.
-add 50 mL to 250 mL LB/amp, incubate 2-4 hrs.
-add IPTG (to 200-400 uM final conc. Incubate 3-4 hrs.
-harvest and centrifuge culture at 5,000 rpm for 7 min. (pellet can be
frozen at -80C at this point).
-resuspend pellet in GST buffer (10mL for every 300 mL BL21 culture)
-transfer to 50 mL ultracentrifuge tube.
-sonicate: 01, 01, 50% for 1 min, twice. Keep on ice.
-centrifuge at 20,000 rpm for 40 min.
-harvest protein supernatant into 15 mL conical vial.
-add 100 uL glutathione beads.
-incubate, spinning, for 2 hr at 4 C.
-centrifuge at 2,200 rpm for 1 min.
-remove supernatant (most).
-wash with 5 mL GST buffer.
-incubate, spinning, for 5 min. at 4 C. Spin down.
-remove supernatant (leave ~1ml of supernatant).
-using snipped tip, transfer beads to 1.5 mL tube.
-centrifuge at 2,200 rpm for 30 sec.
-wash with 1 mL GST buffer, spinning, 30 sec at 4 C. repeat the washing steps 2 more times.
-centrifuge again.
-remove enough supernatant to result in 50% slurry.
-resuspend beads and take 10 ul for coomassie stain.
-use the remaining beads for GST pulldown.
GST buffer: 150 mM NaCl 20 mM Tris pH 7.4 0.5% Triton 0.1 % EDTA filter sterilize.
IPTG: 0.95g in 20 ml filter sterilize with 0.2mm filter.
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GST buffer (Makes 500mL).
25mL of 1M tris pH 7.4
15mL of 5M NaCl
5mL of 500mM EDTA
5mL of 1x triton
450mL of H2O.
Filter sterilize with the vacuum and store at 4C.
Mammalian cell lysate for pull-down
- Re-suspend pellet in GST buffer (5x volume of pellet)
- Sonicate at 01, 01, 50% for 1 min.
- Let it sit in ice for 15 min.
- Spin down at 12000 RPM for 15 minutes.
- Set 100 uL aside of the supernatant for the input.
- Put rest of clear supernatant into a 15ml conical vial. To this vial add the purified GST.
- Incubate overnight/5hrs.
- Spin down at 2000rpm for 30 seconds at 4C
- Remove most supernatant, wash with 5ml of GST buffer, gently resuspend.
- Incubate for 5 min (spinning at 4C)
- Spin down one last time
- Remove ALL supernatant.
- Beads are done (volume should be ~30ml)
- Add 2x for western blot.
Repeat 3x
