Flag-tag or His-tag protein purification
Cell culture
1) Inoculate bacterial cell with 4ml LB for 8~9 hr @37C
2) Transfer to 25ml LB and keep @37C for O/N
3) Transfer to 500ml and keep growing for 1hour.
4) After 1hour, add IPTG (final concentration=0.2mM) and keep growing for
another 4hrs
5) Harvest @5000rpm for 10min
Cell lysis
6) Resuspend the pellet in 14ml BC300 (+PI, DTT, PMSF)
 Transfer to 15ml falcon tube, and then add 140ul of 10% NP40(final 0.1%),
70ul of Na-deoxycholate(final 0.5%), and 70ul of 100mg/ml lysozyme
 Divide into 2X tubes
7) Sonicate @50% for 30sec X 3 times
8) Transfer to 30ml Beckman tube and spin @13000rpm, 4C for 15min.
 Maximum spinning speed for Falcon tube is 8000rpm. That’s why we have to
use Beckman Tube
9) Take supernatant only (7ml X 2 tubes)
Binding with M2-agarose or Nickel-slurry
10) Add same volume of BC0 (+PI, PTT, PMSF)
 final concentration would be around BC150
11) Add 300ul of M2-agarose or Nickel-slurry  incubate @4C for O/N
Washing
12) Spin @8000rpm for 5min.  remove supernatant.
13) Add 5ml of BC250 (+ 0.1%NP40) and rotate @4C for 5min.  1st washing
14) Spin @8000rpm for 5min.  remove supernatant.
15) Add 5ml of BC250 (+ 0.1%NP40) and rotate @4C for 5min.  2nd washing
16) Spin @8000rpm for 5min.  remove supernatant and transfer to 1.5ml
microcentrifuge tube.
17) Add 1ml of BC250 (+ 0.1%NP40) and rotate @4C for 5min.  3rd washing
18) Spin @8000rpm for 1min  remove supernatant.
19) Add 1ml of BC250 (+ 0.1%NP40) and rotate @4C for 5min.  4th washing
20) Spin @8000rpm for 1min  remove supernatant.
Elution
21) Add flag-peptide or 100mM immidazole in 250ul of BC100(+ 0.03% NP40)
22) Incubate @4C for 1hr  meanwhile, vortex several times
23) Spin @8000rpm for 1min and take supernatant  1st elution
24) Add flag-peptide or 200mM immidazole in 250ul of BC100(+ 0.03% NP40)
25) Incubate @4C for 1hr  meanwhile, vortex several times
26) Spin @8000rpm for 1min and take supernatant  2nd elution
27) Add flag-peptide or 200mM immidazole in 250ul of BC100(+ 0.03% NP40)
28) Incubate @4C for 1hr  meanwhile, vortex several times
29) Spin @8000rpm for 1min and take supernatant  3rd elution
30) Check the concentration with BSA standard on SDS-PAGE gel