Purification of T4 PNK
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Purification of T4 Polynucleotide Kinase
[edit]Clone #:
Host;
1. Expression
[edit]2. Capture and Purification
[edit]Ni
3. Purification using Capto SP ImpRes
[edit]- Dilute protein from Ni column with 10 vol of 50 mM NaCl, 20 mM potassium phosphate buffer, pH 7.0
- Load to 20 ml Q-FF (1.6 cm id) coupled with 25 ml Capto SP ImpRes (1.6 cm id) using super-loop
- Wash with 150 ml of 50 mM NaCl, 20 mM potassium phosphate buffer, pH 7.0
- Reverse Q-FF column.
- Linear gradient from 50 mM NaCl to 1.5M NaCl, in 20 mM potassium phosphate buffer, pH 7.0, flow rate 1.5 ml/min, 400 ml gradient, 10 ml/fraction
T4PNK eluted at 15% B, 220m M NaCl
| Lane | Desc | Note | |
| 1,2 | PNK from Ni column, step gradient | Lot 160-1764 |
|
| 3,4, 5 | PNK from NI column, step gradient, then GE haparin column | Lot 160-1764 |
|
| 6,7 | Cre from NI, then QFF-SP FF. | Lot 161-1010 |
|
| 8,9 | Cre from Ni, QFF-SP FF, then GE haparin column | Lot 161-1010 |
