Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

Proteolytic Fragments of Ca 2+ -Binding Proteins 187
2.3. Thrombic Digestion
Fig. 1. (continued) (D) TM1. (E) TM2.
1. 100 mM Tris-HCl, pH 8.5. Can be stored for months at 4°C. Chelex-treated
50 mM NH 4HCO 3. To remove all metal ions, stir buffer using a magnetic
stirrer with approx 5 mL Chelex-100 resin per liter of buffer for about 5 min,
then filter off the resin. Can be stored for weeks at 4°C.
2. Thrombin (Sigma), from bovine plasma. Should be purchased free of all other
clotting factors and proteolytic activities. Reconstitute according to the package
directions or dissolve in an appropriate buffer (e.g., sodium citrate) to a concentration
of 1 U/mL. This enzyme is intolerant to freeze-thawing; dispense into
aliquots of 50–100 U and store at –70°C.
3. Sephadex G-50 (Pharmacia) (100 cm × 1.5 cm) equilibrated with chelexed 50 mM
NH 4HCO 3.
3. Methods
3.1. Tryptic Digestion of Calmodulin
1. Pre-equilibrate a Sephadex G-50 column (100 × 1.5 cm) with buffer A at approx
0.35 mL/min overnight.
2. Dissolve 100 mg CaM in 4 mL freshly prepared buffer A (to approx 1.5 mM CaM
concentration). Add five equivalents CaCl 2 (to approx 7.5 mM; add 15 mL of a 2 M
stock). Take a sample for SDS-PAGE.