Tandem Affinity Purification (TAP) Protocol (Page 2)

Calmodulin-Binding Buffer (CBB) (10 mL)

Final Concentration Volume Stock
25 mM Tris-HCl, pH 8.0 0.25 mL 1 M
150 mM NaCl 0.30 mL 5 M
1 mM Mg acetate 10 µL 1 M
1 mM Imidazole 10 µL 1 M
2 mM CaCl2 20 µL 1 M
10 mM β-mercaptoethanol 7µL 14.3 M
Add Milli-Q water to 10 mL.
Split into a 9 mL and a 1 mL aliquot
Add 90 µL 10% NP-40 to one aliquot to make 0.1% NP-40.
Add 100 µL 10% NP-40 to the other to make 0.02% NP-40.

Calmodulin Elution Buffer (CEB) (2.5 mL)

Final Concentration Volume Stock
25 mM Tris-HCl, pH 8.0 62.5 µL 1 M
150 mM NaCl 75 µL 5 M
0.02% NP-40 5 µL 10%
1 mM Mg acetate 2.5 µL 1M
1 mM Imidazole 2.5 µL 1 M
20 mM EGTA 500 µL 100 mM
10 mM β-mercaptoethanol 1.8 µL 14.3 M
Milli-Q water to 2.5 mL

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