Bioanalytical Research
Bioanalytical Research Group
iTAP Based Protein Complex Purification
Buffers & Reagents
Reagents:
IgG-Sepharose. Fast Flow. Amersham-Pharmacia (17-0969-01) Calmodulin Affinity Resin. Stratagene (214303-52) TEV protease (home-made)
Buffer A
Buffer B
10 mM HEPES, pH 7.9 (1 ml of 1 M stock)
1.5 mM MgCl2 (150 µl of 1 M stock)
10 mM KCl (0.5 ml of 2 M stock)
0.5 mM DTT (50 µl of 1 M stock)
0.5 mM EDTA (100 µl of 0.5 M stock)
1 % Protease Inhibitor Cocktail
H2O to 100 ml final
20 mM HEPES, pH 7.9 (2 ml of 1 M stock)
20 % Glycerol (23 ml of 87 % stock)
200 mM KCl (10 ml of 2 M stock)
0.5 mM DTT (50 µl of 1 M stock)
0.5 mM EDTA (100 µl of 0.5 M stock)
1 % Protease Inhibitor Cocktail
0.5 % NP40 (2.5 ml of 20 % stock)
H20 to 100 ml final
Buffer C
TEV Cleavage Buffer
(for washing)
20 mM HEPES, pH 7.9 (2 ml of 1 M stock)
20 % Glycerol (23 ml of 87 % stock)
200 mM KCl (10 ml of 2 M stock)
0.5 mM DTT (50 µl of 1 M stock)
0.5 mM EDTA (100 µl of 0.5 M stock)
0.1 % NP40 (2.5 ml of 20 % stock)
H20 to 100 ml final
10 mM Tris-Cl, pH 8 (0.5 ml of 2 M stock)
150 mM NaCl (3 ml of 5 M stock)
0.1 % NP40 (0.5 ml of 20 % stock)
0.5 mM DTT (50 µl of 1 M stock)
0.5 mM EDTA (100 µl of 0.5 M stock)
H20 to 100 ml final
Calmodulin Binding Buffer
10 mM Tris-Cl, pH 8 (0.5 ml of 2 M stock)
10 mM beta-mercaptoethanol (69.7 µl of stock)
150 mM NaCl (3 ml of 5 M stock)
1 mM Mg-acetate (100 µl of 1 M stock)
1 mM Imidazole (100 µl of 1 M stock)
2 mM CaCl2 (200 µl of 1 M stock)
0.1 % NP40 (0.5 ml of 20 % stock)
H20 to 100 ml final
Notes:
- Add DTT just before use.
- Binding of endogenous Calmodulin to the Calmodulin binding peptide of the TAP-tag might reduce the binding efficience to the Calmodulin beads. To avoid this effect, it is recommended to add 2 mM EGTA to the Buffers A and B.
- If you are pooling supernatants A and B (see also Tandem affinity purification), adjust the concentration of KCl of -Buffer A to 200 mM KCl just before pooling.