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. 20 mM imidazole pH7.0, 1 mM PMSF (PMSF freshly added, TOXIC!).
(PMSF has a short half-life in aqueous solutions). (Imidazole prevents binding of unspecific proteins to beads).
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.
Option 1, batch purification:
For each prep aliquot 1.5 ml Ni-agarose slurry and wash 2 times with 10 ml TKET (1,000 rpm, 1 min). (The amount of Ni-agarose may have to be titrated).
Add supernatant from sonicated cells to washed Ni-agarose 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 w. 20 mM imidazole pH7.0.
After final wash, remove most of the buffer.
For elution, add 1 ml TKET w. 200 mM imidazole pH7.0.
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.
Option 2, column purification:
Add 2 ml Ni-NTA agarose slurry (=1ml beads) to column.
Let column settle, and storage buffer elute.
Wash with 10 ml TKET w. 20 mM imidazole.
Run supernatant from sonicated cells over column w/o disturbing column material. (optional: reapply flow-through 1-2x).
Wash column three times with 10 ml TKET w. 20 mM imidazole (let everything drip through before applying new buffer).
Elute with one column volume of TKET w. 200 mM imidazole. 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.
Ni-NTA agarose (Qiagen) capacity: 5-10 mg protein per ml beads.
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