Purification of Peroxidase
|
| Column: |
TSKgel Tresyl-5PW, 10 µm,
6 mm ID × 4 cm, modified with concanavalin A |
| Binding: |
0.05 mol/L acetate buffer, pH 5.0 containing 0.5 mol/L NaCl, and 1 mmol/L each of CaCl2, MnCl2, and MgCl2 |
| Mobile phase: |
step gradient at 4 min (see arrow on diagram) to 25 mmol/L α-methyl-D-glucoside in binding buffer |
| Flow rate: |
1.0 mL/min |
| Detection: |
UV @ 403 nm |
| Sample: |
crude peroxidase, 0.5 mg |
| Washing step: |
wash TSKgel Tresyl-5PW, 6 mm ID ×
4 cm, with dissolved H2O |
| Ligand solution: |
Dissolve 40 mg of concanavalin A in 10 mL of 0.1 mol/L NaHCO3 , pH 8.0, containing 0.5 mol/L NaCl
|
| Coupling step: |
recycle the ligand solution overnight through the column at 0.2 mL/min at
25 °C |
| Blocking step: |
block the residual tresyl groups with 0.1 mol/L Tris-HCl, pH 8.0, at 1.0 mL/min for 1 hr at 25 °C |
|
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