Flag-Tag Or His-Tag Protein Purification

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)

à 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

à Divide into 2X tubes

7) Sonicate @50% for 30sec X 3 times

8) Transfer to 30ml Beckman tube and spin @13000rpm, 4C for 15min.

à 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)

à final concentration would be around BC150

11) Add 300ul of M2-agarose or Nickel-slurry à incubate @4C for O/N

Washing

12) Spin @8000rpm for 5min. à remove supernatant.

13) Add 5ml of BC250 (+ 0.1%NP40) and rotate @4C for 5min. à 1st washing

14) Spin @8000rpm for 5min. à remove supernatant.

15) Add 5ml of BC250 (+ 0.1%NP40) and rotate @4C for 5min. à 2nd washing

16) Spin @8000rpm for 5min. à remove supernatant and transfer to 1.5ml microcentrifuge tube.

17) Add 1ml of BC250 (+ 0.1%NP40) and rotate @4C for 5min. à 3rd washing

18) Spin @8000rpm for 1min à remove supernatant.

19) Add 1ml of BC250 (+ 0.1%NP40) and rotate @4C for 5min. à 4th washing

20) Spin @8000rpm for 1min à remove supernatant.

Elution

21) Add flag-peptide or 100mM immidazole in 250ul of BC100(+ 0.03% NP40)

22) Incubate @4C for 1hr à meanwhile, vortex several times

23) Spin @8000rpm for 1min and take supernatant à 1st elution

24) Add flag-peptide or 200mM immidazole in 250ul of BC100(+ 0.03% NP40)

25) Incubate @4C for 1hr à meanwhile, vortex several times

26) Spin @8000rpm for 1min and take supernatant à 2nd elution

27) Add flag-peptide or 200mM immidazole in 250ul of BC100(+ 0.03% NP40)

28) Incubate @4C for 1hr à meanwhile, vortex several times

29) Spin @8000rpm for 1min and take supernatant à 3rd elution

30) Check the concentration with BSA standard on SDS-PAGE gel