Transformation:
Day 1:
Transform expression plasmid into BL21/DE3 pRI952.
Plate on LBA (Amp) plates.

Growth and induction:
Day 2:
Start 10 ml over night culture in LB w. 100 µg/ml Amp, 50 µg/ml Chloramp.

Day 3:
Dilute culture 1:100 into 1000 ml LB w. 100 µg/ml Amp, 50 µg/ml Chloramp. (prewarmed is better).
Follow A600.
At A600 between 0.4-0.5 (do not overgrow!!) transfer culture to 15C.
After 10-15 min, add IPTG to 0.2 mM final concentration.
Grow over night at 15C.
(Temperature, time and IPTG concentration may have to be optimized for individual proteins).

Purification:
Day 4:
Pellet cells at 5,000 rpm, 8 min.
Resuspend cells completely (!) in 25 ml ice cold TKET w. 1 mM PMSF (PMSF freshly added, TOXIC!).
(PMSF has a short half-life in aqueous solutions).
Sonicate 8x30'' with 30'' intervals at 4.5 amplitude on ice.
Add Triton X-100 to 0.5% final.
Save 50 µl for analysis.
Nutate at 4C for 15 min.
Spin 11,000 rpm, 15 min.
For each prep aliquot 1.5 ml Glutathione Sepharose slurry and wash 2 times with 10 ml TKET (1,000 rpm, 1 min). (The amount of Glutathione Sepharose may have to be titrated).
Add supernatant from sonicated cells to washed Glutathione Sepharose beads. (Supernatant should be yellow, avoid pellet!).
Nutate 1-2 hours at 4C.
Spin 1,000 rpm 1 min.
Remove and save supernatant. (Transfer 50 µl to tube for analysis, store remainder at -80C in case more protein needs purified).
Wash beads 5 times with 10 ml TKET.
After final wash, remove most of the buffer.
Elution:
Add 1 ml 100 mM Tris-HCl pH7.5, 100 mM KCl, 20 mM glutathione.
Nutate at RT, 15 min.
Spin 1,000 rpm, 1 min.
Transfer supernatant to tube. Test protein concentration by Bradford (below).
Redo elution until little protein comes off beads (usually 2-3 times).
Pool eluates and dialyse against PBS or other buffer at 4C.

Day 5:
Aliquot dialysed proteins to tubes.
Measure concentration by Bradford and analyse 2-4 µg on SDS-PAGE.
Bradford:
Mix
800 µl water
200 µl Bio-Rad protein assay concentrate
2-20 µg protein
Leave at RT 5 min
Measure A595 (use standard w/o protein)
Compare to a BSA titration from 2-20 µg

Buffers:
TKET:

10 mM Tris-HCl pH 7.5
100 mM KCl
0.1 mM EDTA
0.05 % Triton X100