Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

328 Kobayashi
3.2. Coupling of Cromolyn to AF-amino Toyopearl 650M
1. Dissolve 0.5 µmol (234 mg) of Cromolyn (free form) in 26 mL of a coupling
solution (DMF: H2O = 25 mL: 1 mL) at 40°C. Adjust pH to 4.0 with the dropwise
addition of 1 N HCl. The pH should be measured using pH paper.
2. Wash 5 g (wet weight) of AF-amino Toyopearl with 100 mL of the coupling
solution on a sintered glass filter funnel.
3. Mix the washed resin and the Cromolyn solution with gentle shaking and then
add 0.5 g of EDC in solid.
4. Incubate the mixture with a shaker in a water bath at 40°C for 24 h.
5. After the coupling is completed, wash away excess Cromolyn with 200 mL of the
coupling buffer and 100 mL of distilled water on a sintered glass filter funnel.
6. Block any remaining active group as described in Subheading 3.1., steps 6–8.
3.3. Coupling of Tranilast to AF-amino Toyopearl 650M
1. Dissolve 2.5 µmol (820 mg) of Tranilast in 20 mL of 90% DMF at room temperature.
Adjust pH to 4.5 by the dropwise addition of 1 N HCl. The pH should be
measured using pH paper.
2. Wash 5 g (wet weight) of AF-amino Toyopearl with 100 mL of 90% DMF (pH
4.5) on a sintered glass filter funnel.
3. Mix the washed resin and the Tranilast solution and then add 1 g of EDC in solid.
4. Incubate the mixture for 1 h with gentle shaking and readjust the pH to 4.5 by 1 N
NaOH or 1 N HCl, and further shaken for 24 h at room temperature.
5. Wash the resin successively with 100 mL of 90% DMF (without adjusting pH)
and 100 mL of distilled water.
6. Block any remaining active group as described in Subheading 3.1., steps 6–8.
3.4. Coupling of Fluphenazine to Epoxy-Activated Sepharose 6B
1. Dissolve 200 µmole (102 mg) of Fluphenazine·2HCl in 4.9 mL of a coupling
solution (Dioxane: H 2O: 1 N NaOH = 2 mL: 2 mL: 0.9 mL, pH 10.0).
2. Weight out 0.5 g Epoxy-activated Sepharose 6B (0.5 g freeze-dried powder gives
about 1.7 mL of gel) and suspend it in 50 mL of distilled water. Wash swollen gel
with 200 mL of distilled water and 20 mL of the coupling buffer on a sintered
glass filter funnel.
3. Mix the washed gel and the Fluphenazine solution and incubate with gentle shaking
in a water bath at 45°C for 24 h.
4. After the coupling is completed, wash the gel with 100 mL of the coupling solution,
pH 10.0, on a sintered glass filter funnel.
5. Any nonreacted groups on the gel should be blocked with gentle shaking in 1 M
ethanolamine, pH 8.0, for 15 h at room temperature.
6. Wash the product successively with 100 mL of distilled water, and 100 mL of
rinsing buffers A and B on a sintered glass filter funnel. The coupled affinity resin
is now ready for use.