2.5 Desalting Step
1. 5 mL HiTrap Desalting column prepacked with Sephadex
G-25 resin (GE Healthcare).
2. AKTA protein purification system or equivalent.
3. Prepare 100 mL CBP-binding buffer: 50 mM HEPES-KOH
pH 8.0 (5 mL of 1 M stock solution), 130 mM NaCl (2.6 mL
of 5 M stock solution), 10% glycerol, 2 mM CaCl 2 (200 μL of
1 M stock solution), 0.03% DDM (0.3 mL of 10% stock
solution).
2.6 Calmodulin
Affinity
Chromatography
1. Calmodulin affinity resin (Agilent Technology).
2. Gravity flow column (e.g., MoBiTec, #10141).
3. Prepare 100 mL CBP-binding buffer: 50 mM HEPES-KOH
pH 8.0 (5 mL of 1 M stock solution), 130 mM NaCl (2.6 mL
of 5 M stock solution), 10% glycerol, 2 mM CaCl 2 (200 μL of
1 M stock solution), 0.03% DDM (0.3 mL of 10% stock
solution).
4. Prepare 100 mL CBP washing buffer: 50 mM HEPES-KOH
pH 8.0 (5 mL of 1 M stock solution), 130 mM NaCl (2.6 mL
of 5 M stock solution), 10% glycerol, 0.2 mM CaCl 2 (20 μL of
1 M stock solution), 0.03% DDM (0.3 mL of 10% stock
solution).
5. Prepare 25 mL CBP elution buffer: 50 mM HEPES-KOH
pH 8.0 (1.25 mL of 1 M stock solution), 400 mM NaCl
(2 mL of 5 M stock solution), 3% glycerol, 2 mM EGTA
(100 μL of 0.5 M stock solution), 0.03% DDM (0.3 mL of
10% stock solution).
6. NanoDrop spectrophotometer (Thermo Scientific).
2.7 Sample
Concentration
1. Amicon Ultra centrifugal filter units, molecular weight cut-off
100 kDa, Ultra-15 (Amicon).
2. Centrifuge, e.g., Heraeus Megafuge 16R.
3. Prepare 10 mL S6 buffer: 20 mM HEPES-KOH pH 8.0
(0.2 mL of 1 M stock solution), 130 mM NaCl (0.26 mL of
5 M stock solution), 5 mM MgOAc 2 (50 μL of 1 M stock
solution), 0.03% DDM (30 μL of 10% stock solution).
2.8 Size Exclusion
Chromatography
1. Superose 6 Increase 10/300 GL column (GE Healthcare).
2. AKTA protein purification system or equivalent.
3. Prepare 250 mL S6 buffer: 20 mM HEPES-KOH pH 8.0
(5 mL of 1 M stock solution), 130 mM NaCl (6.5 mL of
5 M stock solution), 5 mM MgOAc 2 (1.25 mL of 1 M stock
solution), 0.03% DDM (0.75 mL of 10% stock solution).
10
Burak V. Kabasakal et al.
1. 5 mL HiTrap Desalting column prepacked with Sephadex
G-25 resin (GE Healthcare).
2. AKTA protein purification system or equivalent.
3. Prepare 100 mL CBP-binding buffer: 50 mM HEPES-KOH
pH 8.0 (5 mL of 1 M stock solution), 130 mM NaCl (2.6 mL
of 5 M stock solution), 10% glycerol, 2 mM CaCl 2 (200 μL of
1 M stock solution), 0.03% DDM (0.3 mL of 10% stock
solution).
2.6 Calmodulin
Affinity
Chromatography
1. Calmodulin affinity resin (Agilent Technology).
2. Gravity flow column (e.g., MoBiTec, #10141).
3. Prepare 100 mL CBP-binding buffer: 50 mM HEPES-KOH
pH 8.0 (5 mL of 1 M stock solution), 130 mM NaCl (2.6 mL
of 5 M stock solution), 10% glycerol, 2 mM CaCl 2 (200 μL of
1 M stock solution), 0.03% DDM (0.3 mL of 10% stock
solution).
4. Prepare 100 mL CBP washing buffer: 50 mM HEPES-KOH
pH 8.0 (5 mL of 1 M stock solution), 130 mM NaCl (2.6 mL
of 5 M stock solution), 10% glycerol, 0.2 mM CaCl 2 (20 μL of
1 M stock solution), 0.03% DDM (0.3 mL of 10% stock
solution).
5. Prepare 25 mL CBP elution buffer: 50 mM HEPES-KOH
pH 8.0 (1.25 mL of 1 M stock solution), 400 mM NaCl
(2 mL of 5 M stock solution), 3% glycerol, 2 mM EGTA
(100 μL of 0.5 M stock solution), 0.03% DDM (0.3 mL of
10% stock solution).
6. NanoDrop spectrophotometer (Thermo Scientific).
2.7 Sample
Concentration
1. Amicon Ultra centrifugal filter units, molecular weight cut-off
100 kDa, Ultra-15 (Amicon).
2. Centrifuge, e.g., Heraeus Megafuge 16R.
3. Prepare 10 mL S6 buffer: 20 mM HEPES-KOH pH 8.0
(0.2 mL of 1 M stock solution), 130 mM NaCl (0.26 mL of
5 M stock solution), 5 mM MgOAc 2 (50 μL of 1 M stock
solution), 0.03% DDM (30 μL of 10% stock solution).
2.8 Size Exclusion
Chromatography
1. Superose 6 Increase 10/300 GL column (GE Healthcare).
2. AKTA protein purification system or equivalent.
3. Prepare 250 mL S6 buffer: 20 mM HEPES-KOH pH 8.0
(5 mL of 1 M stock solution), 130 mM NaCl (6.5 mL of
5 M stock solution), 5 mM MgOAc 2 (1.25 mL of 1 M stock
solution), 0.03% DDM (0.75 mL of 10% stock solution).
10
Burak V. Kabasakal et al.