Reference: Smith and Johnson, Gene 67, 31-40 (1989); Smith et al., PNAS
83:8703.
Date of this version: 11/4/96 (by Genhong Cheng)
Reagents:
#1. Growth medium
LB ampicillin (100 µg/ml)
#2. 1000x IPTG
100 mM in distilled water (0.238g in 10 ml H2O), stored at -20 C.
#3. lysis buffer
Reagent [final] stock Volume
EDTA pH 8.0 100 mM 0.5 M 10 ml
Triton X-100 1% 100% 0.5 ml
PBS 1x 1x 38.5 ml
PMSF 1mM 100 mM* 0.5 ml
Aprotinin 1% 100%** 0.5 ml
*100 mM PMSF, 0.174 g in 10 ml of Isopropanol, add just before use
**aprotinin stock solution from Sigma; add just before use
#4. glutathione-agarose obtained from Sigma
#5. PBS
Procedure
1. Grow a 25 ml overnight culture in LB of bacteria with the desired GEX construct.
2. Dilute the overnight (1:20) into 500 ml fresh LB+ and grow at 37 C for 2
hours.
3. Add 0.5 ml 100mM IPTG ( final concentration of 100 µM).
4. Grow another 2-4 hours after addition of IPTG
5. Spin down bacteria (JLA, 4000 rpm x 10 min, 4 C).
6. Drain medium out of bottles as complete as possible.
7. Resuspend the pellets in 10 ml of lysis buffer and transfer the bacteria
into 50 ml
tube.
8. Sonicate 1 minutes at the setting of 4 (keep on ice during sonication; avoid
foaming).
9. Pellet membranes from lysed bacteria (JA-12, 4000 rpm x 10 minutes, 4 C ).
10. Place supernatant in 15 ml conical screwcap tubes; add 0.25 ml bed volume
of glutathione beads and rock gently at 4 C for 1 hour.
11. Wash by spinning down beads (TJ6, set 5 for 5 minutes, 4 C) and resuspending
in 10 ml volumes cold lysis buffer.
12. Repeat 3 X wash with PBS.