Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

S-100 Protein Interacting Drugs 327
Fig. 1. Structures of substances mentioned.
12. Buffer C: 20 mM Tris-HCl, 2 mM CaCl 2, pH 7.5.
13. Buffer D: 20 mM Tris-HCl, 6 M urea, pH 7.5.
3. Methods
3.1. Coupling of Amlexanox to AF-amino Toyopearl 650M
1. Wash the resin (5 g wet weight) with 20 mL of DMF by decantation (three times).
2. Add 0.34 mmol (100 mg) of Amlexanox (dissolved in 1 mL of DMF) to the
washed gel.
3. After gentle mixing, add EDC (0.15 g, suspended in 10 mL of DMF) to the suspension.
Adjust the pH of the mixture to 5.0 by the dropwise addition of 1 N HCl.
The pH should be measured using pH paper because organic solvents may damage
electrodes.
4. After incubation with gentle shaking for 24 h at room temperature, readjust the
pH to 5.0 by the dropwise addition of 1 N NaOH or 1 N HCl and further incubate
for 24 h.
5. Wash the resin successively with 20 mL of DMF (three times) and 50 mL of
distilled water (three times) on a sintered glass filter funnel.
6. Block any remaining active groups by incubating the washed resin with 0.2 M
sodium acetate (4 mL) and acetic anhydride (2 mL) for 30 min at 0°C.
7. Add 2 mL of acetic anhydride to the gel suspension and incubate with gentle
shaking further for 30 min at room temperature.
8. Wash the resin successively with distilled water (100 mL), 0.1 N NaOH (100 mL)
and distilled water (300 mL) with a sintered glass filter funnel. The coupled affinity
resin is now ready for use (see Note 1).