Solutions

1. 50 mM NaOAc, pH 5.2. ~200 mL.
2. 10 mM NaOAc, pH 5.2. ~ 1 L.
3. 1 M ethanolamine/HCl, pH 8.0. ~ 50 mL.

Protocol

For 25 ml Affi-Gel 15 (BioRad), you can couple 100 mg cTnC. The column’s capacity will be ~50 mg of cTnI.

1. Dissolve 100 mg cTnC in 35 mL 50 mM NaOAc at 4 ^oC.
2. Wash 25 mL Affi-Gel-slurry with ~5 vol of cold 10 mM NaOAc for 10 min.
3. Repeat w/ 3 vol of 10 mM NaOAc.
4. Pour the gel into 10 mL beaker and take out extra buffer. The volume should be 25 mL.
5. Add 35 mL cTnC solution. Transfer the mixture to two 50 mL conical tubes.
6. Rock the mixture overnight @ 4 ^oC.
7. Drain out the protein solution and save this, too.
8. Then re-suspend the gel in 25 mL 1M ethanolamine/HCl (pH 8.0). Do step 9 in the hood and leave the gel for 1 hr at room temperature.
9. After 1 hr, drain out the ethanolamine/HCl.
10. Wash the gel with 10-20 vol of cold 10 mM NaOAc twice.
11. Throughout the procedure, do not let the gel dry.