TSKgel Tresyl-5PW
TSKgel Tresyl-5PW is based on a 100 nm (1000 Å) pore size polymethacrylate base material derivatized with a 2,2,2-trifluroethanesulfonyl ligand. Unlike other TSKgel affinity columns, the TSKgel Tresyl-5PW columns require activation with a user-selected ligand containing amino, thiol, phenol, or imidazole groups. The resulting structure is literally a custom affinity ligand with excellent pH stability and minimal ligand loss due to leaching. TSKgel Tresyl-5PW readily reacts with amino or thiol groups to form stable covalent alkylamines or thioethers.
Structure of TSKgel Tresyl-5PW |
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Purification of peroxidase |
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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 a-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 |