GST-Protein Purification
1st day: Bacteria Culture, prepare 50ml LB flask, 200ml LB flask
1) start with 4ml culture @37C for 8~9hrs
2) Transfer culture to 50ml LB flask @37C for O/N
2nd day: Bacterial Culture  inducer, IPTG  Purification
1) Transfer 50ml culture to 200ml LB flask, 37C for 1hr
2) After 1hr, add 250ul of 1M IPTG (1/1000 volume of LB media)  37C for
additional 4~5hr
Cell lysis
3) Spin @5000rpm for 5min
4) Remove supernatant and leave bacterial cell pellet
5) Resuspend bacterial pellet with 7ml of BC300 (DTT, PMSF, Protease inhibitor)
( To make BC300, BC500 : BC0= 3 : 2)
6) After Resuspend, add 10%NP40  final conc. should be 0.1%
10% Na-deoxycholate  final conc. should be 1%
7) Divide into 2 X 15ml falcon tube (about 4ml/each)
8) Add 0.1 volume of 10mg/ml lysozyme. We have 100mg/ml lysozyme, you can
add 0.01 volume (around 40ul/each, final conc. should be 0.5mg~1mg/ml)
9) Sonicate @50% output 1min X 3 times or 40sec X 4 times
10) After sonication, transfer to 30ml of Beckman tube
(Because 15ml falcon tube will be broken @13000rpm)
11) Spin @13000rpm 4C for 15min
Binding with Glutathione-sepharose
12) Collect supernatant only in 15ml falcon tube
13) Add BC0 upto 15ml (final conc. would be around BC150) + DTT, PMSF, PI
14) Add 400ul of 50% Glutathione-sepharose slurry
15) Bind GST protein by rotating for 40min @RT (or 2hr @4C)
16) During ratating, prepare 10ml washing buffer, BC200/prep (Add 10% NP40 to
make final conc. 0.1%) How to make BC200 BC500 : BC0 = 2 : 3
Wahing
17) Before washing, spin @8000rpm for 5min to completely spin down the sepharose
beads
18) Remove supernatant and add 5ml of washing buffer
19) Rotating @RT for 5min
20) Spin @8000rpm for 5min again. Remove 4ml and leave 1ml supernatant to
transfer to 1.5ml tube
21) Transfer 1ml of supernatant and sepharose bead to 1.5ml tube
22) Spin @8000rpm for 1min
23) Remove supernatant and add 1ml washing buffer
24) Rotating @RT for 5min  Repeat twice more for washing
25) Finally, spin @8000rpm for 1min, remove all supernatant and add 250ul of PBS
(+PI, DTT, PMSF)
26) Store @4C and Run SDS-PAGE to check concentration
Buffer Recipe
BC0 (0 means no salt)
20mM TrisHCl pH8.0
20% Glycerol
0.2mM EDTA pH8.0
BC500 (500 means 500mM salt)
20mM TrisHCl pH8.0
20% Glycerol
0.2mM EDTA pH.8.0
500mM NaCl