Preparation of Affinity Column

SEK 5/3/95

1. Add 222.2 µl 1 M MOPS, pH 7.5 to 2 ml of 10 mg of CREBtide (0.1 M MOPS pH 7.5 final concentration).

2. Read OD205, OD280 (using 0.1 M MOPS buffer as blank).

3. Affigel-10 is stored frozen at < –70°C. Thaw at 4°C for ~20 min.

4. Filter 1–2 ml on nylon (0.22 µM pore size), using Buchner funnel and vacuum. Do not allow beads to dry.

5. Wash bed with 3x2 ml dH²O.

6. Scrape 0.5–1 ml into 15 ml microfuge tube.

7. Add 2.22 ml CREBtide ligand solution to tube. Rock at 4°C for 4 h.

8. Transfer slurry to column.

9. Wash 2x500 µl dH²O.

10. Collect eluate and save at –20°C. Read OD.

11. Wash column:

2x5 ml cold PBS

1x5 ml PBS/0.02% NaN3 (1 µl NaN3 in 5 ml PBS)

12. Store at 4°C upright in beaker in PBS/NaN3 + parafilm.