Preparation of Affinity Column

 

SEK 5/3/95

 

 

1. Add 222.2 l 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 < 70C. Thaw at 4C for ~20 min.

 

4. Filter 12 ml on nylon (0.22 M pore size), using Buchner funnel and vacuum. Do not allow beads to dry.

 

5. Wash bed with 3x2 ml dH2O.

 

6. Scrape 0.51 ml into 15 ml microfuge tube.

 

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

 

8. Transfer slurry to column.

 

9. Wash 2x500 l dH2O.

 

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

 

11. Wash column:

2x5 ml cold PBS

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

 

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