Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

342 Walsh et al.
11. Bovine serum albumin (BSA; 10 mg/mL stock in ddH 2O).
12. Purified CaM (100–300 µM stock).
13. Purified NOS (2–4 mg/mL). Stable for months when frozen at –80°C. Avoid
freeze-thawing by storing as small aliquots.
14. All stocks are stable for 6 mo or longer when kept frozen at –20°C, unless otherwise
stated.
2.4. NOS NADPH Oxidation Assay
Same reagents as for the oxyhemoglobin assay, except HbO2, SOD, and BSA
are not required and a more dilute solution of catalase (10,000 U/mL stock in
ddH2O) is used.
2.5. NOS Cyt c Reduction Assay
1. 50 mM HEPES buffer (pH 7.5).
2. CaCl 2 (0.5 M stock in ddH 2O).
3. NADPH (approx 10 mg/mL; 10 mM stock solution in ddH 2O).
4. Cyt c (5 mM stock solution in buffer).
5. Purified CaM (100–300 µM stock).
6. All of the above stocks are stable for 6 mo or longer when kept frozen at –20°C.
7. Purified NOS (2–4 mg/mL). Stable for months when frozen at –80°C. Avoid
freeze-thawing by storing as small aliquots.
2.6. NOS Citrulline Assay
1. Same as for the oxyhemoglobin assay, except a scintillation counter, but no spectrophotometer,
HbO 2, SOD, catalase, or BSA are required.
2. L-[ 14 C]Arg (200 mM stock with a specific activity of approx 3 µCi/µmol). Stable
for months when refrigerated.
3. A strong cation exchange resin such as Bio-Rad 50W-X8. Must be converted to
the sodium form by washing with 5 vol of 1 N NaOH, then neutralized with 5 vol
of water.
2.7. MLCK Assay
1. A scintillation (beta) counter.
2. A temperature-controlled water bath.
3. Purified CaM or CaM mutant stocks aforementioned.
4. Purified substrate: LC 20 (the 20 kDa light chain of myosin II). Stable for years
at –80°C.
5. 50 mM Tris-HCl (pH 7.5), 120 mM KCl, 8 mM MgCl 2, 0.2 mM CaCl 2, 2 mM
DTT, 0.2% (v/v) Tween-80.
6. Purified MLCK (0.2 mg/mL). Stable for years at –80°C.
7. [γ- 32 P]ATP (>5000 Ci/mmol). Stock solution of 6 mM with a specific activity of
150–200 cpm/pmol.
8. P81 phosphocellulose paper (Whatman).