MS 3 14 2017
MS 3 14 2017
[edit]1. Grow 2409 iFLP in LB/K/C
Expression
[edit]1. Take out 2409 protein expression stock and inoculate 200 ml LB//Kan/Chl.
2. Grow at 37 ºC o/n.
3. Split the overnight culture into 8 x 1L LB medium, add 1 ml of 10,000X Kan and 1 ml of 10,000X Chl, 20 ml of 20X50 solution.
4. Grow at 37 ºC 2~4 hours.
5. When cell density OD600 is ~0.8, add 0.5 ml of 1 M IPTG to 1 L culture and induce at 25 ºC(Winter 25ºC, Summer 20ºC) O/N.
6. In the morning, spin down cells, combine. Weigh cell pellet, record weight is 40.67g. Store cell pellet at -20 ºC.
Purification
[edit]1. Add 800 ml Buffer A to cell pellet, resuspend cell pellet using a spatula
2. Homogenize the cell suspension with a homogenizer.
3. 2409 iFLP is not able to incubate at 75 ºC.
4. Spin at 6000 rpm for 15 min at 4 oC using high speed centrifuge, collect supernatant.
5. Add cold 400 ml Buffer A to pellet. Homogenize again.
6. Spin at 6000 rpm for 15 min at 4 oC using high speed centrifuge, collect supernatant.
7. Transfer supernatant to ultracentrifuge tubes. Cool ultraspeed rotor with dry ice for 5 min.
8. Spin at 25,000 rpm for 30 min at 4 oC using ultra speed centrifuge, collect clarified supernatant.
9. Load supernatant to 20 ml Ni column in cold room, flow rate ~ 1 ml/min
10. Wash Ni column with 200~500 ml Ni Washing Buffer (Buffer A). Use Buffer A as blank, measure A280.
11. Wash Ni column with 50 ml 20 mM Tris-HCl Buffer, pH 8.0.
12. Elute Taq enzyme with ~200 ml Buffer B (low salt, concentration of NaCl is 100mM), Collect fractions 5 ml/tube using a fraction collector.
13.Use Buffer B as blank, measure protein concentration in each fraction. Identify protein peak, combine fractions with high A280 value (3-11). (A280>0.05)
| Num |
A280(mg/mL) |
Num |
A280(mg/mL) |
Num |
A280(mg/mL) |
| 1 |
0.14 |
6 |
1.09 |
11 |
0.34 |
| 2 |
0.13 |
7 |
0.82 |
12 |
0.25 |
| 3 |
0.53 |
8 |
0.60 |
13 |
0.19 |
| 4 |
2.72 |
9 |
0.44 |
14 |
0.16 |
| 5 |
1.41 |
10 |
0.40 |
15 |
0.15 |
14. Add 1 volume of cold Dilution Buffer.
15. Load Taq onto a 20~25 ml Q HP column (1.6 X10) using a peristaltic pump. With speed 15.
16. Use Dilution Buffer as blank, measure A280 of sample, A280=0.03.
17. Wash column with 100 ml Tris-HCl Buffer, flow rate 2 ml/min
18. Elute Taq using 200 ml of linear gradient of
A: Q HP Washing Buffer
B: Q HP Elution Buffer
Flow rate 1.5 or 2 ml/min.
After elution, set the target 50%--> 100%, 20ml; then set the target 100%--> 0, 50ml.
Use ddH2O as blank, measure A280.
| 18 |
0.02 |
| 19 |
0.01 |
| 20 |
0.08 |
| 21 |
0.06 |
| 22 |
0.27 |
| 23 |
0.55 |
| 24 |
0.89 |
| 25 |
1.44 |
| 26 |
2.56 |
| 27 |
1.83 |
| 28 |
1.48 |
| 29 |
0.61 |
| 30 |
0.45 |
| 31 |
0.58 |
| 32 |
0.31 |
| 33 |
0.11 |
| 34 |
0.06 |
| 35 |
0.05 |
| 36 |
0.06 |
| 40 |
0.09 |
19. Run SDS-PAGE gel on the first peak. If there is anothe peak at ~600~800 mM KCl, it could be DNA/RNA peak, discard the DNA/RNA peak.
| Number |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
11 |
12 |
| sample |
MW |
22 |
23 |
24 |
25 |
26 |
27 |
28 |
29 |
30 |
31 |
32 |
| uL |
10 |
4 |
2 |
2 |
1 |
1 |
1 |
1 |
2 |
3 |
2 |
4 |
| ddH2O uL |
\ |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
| buffer uL |
\ |
4 |
4 |
4 |
4 |
4 |
4 |
4 |
4 |
4 |
4 |
4 |
20. Combine fractions containing Taq (only26).
21. Use ddH2O wash the gel. Boil the gel in microwave for 2 min, two times.
22. Use N3-EF wash Buffer to wash the gel in microwave for 2 min.
23. Use ddH2O wash the gel in microwave for 2 min, two times.
24. Dialysis. Add combined fractions into dialysis bag. Use Buffer B (High salt) to wash the tube and add into bag.
25. Collect the liquid in Dialysis bag, use Dialysis Buffer (storage buffer) to wash bag, 1mL per time, twice, add into collect tube.
26. Measure A280=0.63 mg/ml, total 10.8 ml.
27. Use Dialysis Buffer to wash bag, 1mL per time, until A280 is low. #1 0.59mg/ml, #2 0.53mg/ml
28. Store at -80 oC.
QC
[edit]| 1 |
2 |
3 |
4 |
5 | |
| temp |
0.1 |
0.1 |
0.1 |
0.1 |
0.1 |
| dNTP |
1 |
1 |
1 |
1 |
1 |
| F |
1 |
1 |
1 |
1 |
1 |
| R |
1 |
1 |
1 |
1 |
1 |
| Buffer |
2 |
2 |
2 |
2 |
2 |
| Enzyme 1ul |
positive |
0.1mg/ml |
10ug/ml |
1ug/ml |
0.1ug/ml |
| ddH2O |
3.9 |
3.9 |
3.9 |
3.9 |
3.9 |
0.1ug/ml is good enough.
Regeneration of Ni column
[edit]1. Wash Ni column with 5 CV of water.
2. Wash column with 3 CV of 100 mM EDTA, 500 mM NaCl.
3. Wash the column with 5 CV of H2O.
4. Recharge the column with 1 column volume of 100 mM NiSO4, or NiCl2.
5. Wash the column with 10 column volumes of H2O.
6. Equilibrate with 3 volumes of a suitable buffer (e.g., Buffer A).
Buffer A
20 mM Tris-HCl, pH 7.9~8.0
500 mM NaCl
0.1 mM EDTA
0.1% Tween 20
0.1% Nonident-P40
Ni Washing Buffer
20 mM Tris-HCl, pH 7.9~8.0
800 mM NaCl
10 mM imidazole
Tris-HCl Buffer
20 mM Tris-HCl, pH 7.9~8.0
Buffer B (Low Salt)
20 mM Tris-HCl, pH 7.9~8.0
100 mM NaCl
150 mM imidazole
5% glycerol
Dilution Buffer
20 mM Tris-HCl, pH 7.9~8.0, optional: 5% glycerol
Q HP Washing Buffer
20 mM Tris-HCl, pH 7.9~8.0
50 mM KCl
5% Trehalose
Q HP Elution Buffer
20 mM Tris-HCl, pH 7.9~8.0
1 M KCl
5% Trehalose
Buffer E
20 mM HEPES (pH 8.3) or Tris-HCl, pH 8.0
Buffer F
20 mM HEPES (pH 8.0) or Tris-HCl, pH 7.9
50 mM KCl.
Buffer G
20 mM HEPES or Tris-HCl, pH 7.9 or pH 8.0
500 mM KCl.
Storage Buffer
20 mM HEPES, pH 7.5
100 mM KCl
0.1 mM EDTA
1 mM dithiothreitol
50% glycerol.