Purification of His Tag Protein
Transformation
[edit]1. Transform BL21 (DE3) pLysS with plasmid DNA
2. Grow on LB/Kan/Chl or LB/Amp/Chl plate o/n at 37 Oc
3. Pick a few colonies, grow in 15 ml LB/Kan/Chl or LB/Amp/Chl. Spin down cells to make glycerol stock, Store at -80 oC
Expression
[edit]1. Take out glycerol stock and inoculate 200~500 ml LB//Kan/Chl or LB/Amp/Chl
2. Grow at 37 oC o/n.
3. Add 1 ml of 10,000X Kan or Amp and 1 ml of 10,000X Chl, 20 ml of 20X50 solution to 1 L LB medium to make Autoinduce Medium
4. Split the overnight culture into 8 L Autoinduce Medium, Grow 37 oC 2~4 hours.
5. When cell density OD600 is ~1, add 0.5 ml of 1 M IPTG to 1 L culture and induce at 37 oC or 25 oC O/N.
5. In the morning, spin down cells, combine. Weigh cell pellet, record weight. Store cell pellet at -20 oC
6. Continue to grow cells (8 L per day) for a few more days until total cell pellet is >100 g.
Purification for Acidic Proteins (pI<8.0)
[edit]1. Add 400 ml Buffer A to cell pellet, resuspend cell pellet using a spatula
2. Homogenize the cell suspension with a homogenizer.
Thermostable enzymes:
3. For thermostable enzymes, incubate the homogenate at 75 oC for 30 min, mix ocasionally, cool on ice for 15 min.
4. Spin at 6000 rpm for 15 min at 4 oC using high speed centrifuge, collect supernatant.
5. Add cold 200 ml Buffer A to pellet. Homogenize again.
6. Spin at 6000 rpm for 15 min at 4 oC using high speed centrifuge, collect supernatant.
5. Transfer combined supernatant to ultracentrifuge tubes. Cool ultraspeed rotor with dry ice for 2~5 min.
6. Spin at 25000 rpm for 30 min at 4 oC using ultra speed centrifuge, collect clarified supernatant.
For other enzymes
3. Transfer homogenate to ultracentrifuge tubes. Cool ultraspeed rotor with dry ice for 2~5 min.
4. Spin at 25000 rpm for 30 min at 4 oC using ultra speed centrifuge, collect clarified supernatant
5. Add cold 200 ml Buffer A to pellet. Combine and homogenize again.
6. Spin at 25000 rpm for 30 min at 4 oC using ultra speed centrifuge, collect clarified supernatant
Ni-Column Purification
7. Combined clarified supernatant
8. Load supernatant to 20 ml Ni column in cold room, flow rate ~ 1 ml/min
9. Wash Ni column with 200~500 ml Ni Washing Buffer unitl OD280 ~= 0.1
10. Wash Ni column with 100~200 ml of 1.5 M NaCl-20 mM K2PO4, pH 7.0.
11. Wash Ni column with 50 ml 20 mM Tris-HCl Buffer, pH 8.
12. Elute enzyme with ~200 ml Buffer B, Collect 5 ml fractions using a fraction collector
13. Measure protein concentration in each fraction. Identify protein peak, combine fractions with OD280 >0.05.
14. Add 1 volume of cold Dilution Buffer.
15. Load diluted enzyme onto a 20~25 ml Q HP column (1.6 X10 or 1.6 X20) using a peristaltic pump
16. Wash column with 50 ml Tris-HCl Buffer, flow rate 2 ml/min
17. Elute enzyme 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
17. Check protein concentration in each fractions with protein peak using a nanodrop. Run 1~5 ug protein SDS-PAGE gel on the first major peak. If there is anothe peak at ~600~800 mM NaCl, it could be DNA/RNA peak, discard the DNA/RNA peak.
18. Combine fractions containing desized enzyme.
19. Concentrate enzyme using spin column if concentration is less than 1 mg/ml
20. Dialysis twice against Storage Buffer (80 ml, then 50 ml), change buffer after overnight dialysis.
Purification for Basic Proteins (pI >8.0)
[edit]1. Add 400 ml Buffer A to cell pellet, resuspend cell pellet using a spatula
2. Homogenize the cell suspension with a homogenizer.
Thermostable enzymes:
3. For thermostable enzymes, incubate the homogenate at 75 oC for 30 min, mix ocasionally, cool on ice for 15 min.
4. Spin at 6000 rpm for 15 min at 4 oC using high speed centrifuge, collect supernatant.
5. Add cold 200 ml Buffer A to pellet. Homogenize again.
6. Spin at 6000 rpm for 15 min at 4 oC using high speed centrifuge, collect supernatant.
5. Transfer combined supernatant to ultracentrifuge tubes. Cool ultraspeed rotor with dry ice for 2~5 min.
6. Spin at 25000 rpm for 30 min at 4 oC using ultra speed centrifuge, collect clarified supernatant.
For other enzymes
3. Transfer homogenate to ultracentrifuge tubes. Cool ultraspeed rotor with dry ice for 2~5 min.
4. Spin at 25000 rpm for 30 min at 4 oC using ultra speed centrifuge, collect clarified supernatant
5. Add cold 200 ml Buffer A to pellet. Combine and homogenize again.
6. Spin at 25000 rpm for 30 min at 4 oC using ultra speed centrifuge, collect clarified supernatant
Ni-Column Purification
7. Combined clarified supernatant
8. Load supernatant to 20 ml Ni column in cold room, flow rate ~ 1 ml/min
9. Wash Ni column with 200~500 ml
10. Wash Ni column with 50 ml 20 mM Phosphate Buffer, pH 7.0
11. Elute Taq enzyme with ~200 ml Phospho-Buffer B , collect 5 ml fractions using a fraction collector
17. Measure protein concentration in each fraction. Identify protein peak, combine fractions with OD280 >0.05.
18. Add 1 volume of cold Phospho Dilution Buffer.
19. Load enzyme onto a 20~25 ml SP FF column (1.6 X10 or 1.6 X20) using a peristaltic pump
20. Wash with 50 ml of 20 mM Phosphate Buffer, flow rate 2 ml/min
21. Elute enzyme using 200 ml of linear gradient of
A: SP Washing Buffer
B: SP Elution Buffer
Flow rate 1.5 or 2 ml/min
22. Check protein concentration in each fractions with protein peak using a nanodrop. Run 1~5 ug protein SDS-PAGE gel on the first major peak. If there is anothe peak at ~600~800 mM NaCl, it could be DNA/RNA peak, discard the DNA/RNA peak.
23. Combine fractions containing disired enzyme.
24. Concentrate enzyme using spin column if concentration is less than 1 mg/ml
25. Dialysis twice against Storage Buffer (80 ml, then 50 ml), change buffer after overnight dialysis.
QC
[edit]Make serial dilutions
| Name | Dilution | Concentration | Enzyme | Storage Buffer |
| D1 | 1:x | 1 ug/ul | dilute to 1 mg/ml | Y ul |
| D2 | 1:10 of D1 | 100 ng/ul | D1, 1 ul | 9 ul |
| D3 | 1:100 of D1 | 10ng/ul | D2, 1 ul | 9 ul |
| D4 | 1:1000 of D1 | 1 ng/ul | D3, 1 ul | 9 ul |
| D5 | 1:10000 of D1 | 0.1 ng/ul | D4, 1 ul | 9 ul |
Perform QC assay such as PCR
Record all data on ELN.
Create a new database entry on Protein Database http://www.excellgen.com/protein/index.php
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).
Buffers
[edit]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
Phospho-Buffer B (Low Salt)
20 mM Phosphate, pH 7.0
100 mM NaCl
150 mM imidazole
5% glycerol
Dilution Buffer
20 mM Tris-HCl, pH 7.9~8.0, 5% glycerol
Phospho Dilution Buffer. 20 mM Potassium or Sodium Phophate, pH 7.0, 5% glycerol
Q HP Washing Buffer
20 mM Tris-HCl, pH 7.9~8.0
50 mM KCl
Q HP Elution Buffer
20 mM Tris-HCl, pH 7.9~8.0
0.8 M NaCl
20 mM Phosphate Buffer,
20 mM Potassium or Sodium Phophate, pH 7.0, 5% glycerol
SP Washing Buffer
20 mM Potassium or Sodium Phophate, pH 7.0
50 mM NaCl
SP Elution Buffer
20 mM Potassium or Sodium Phophate, pH 7.0
1 M NaCl
Storage Buffer
20 mM HEPES, pH 7.9
100 mM KCl
0.1 mM EDTA
1 mM dithiothreitol
50% glycerol.