Preparation of Affinity Column
SEK 5/3/95
- Add 222.2ul 1M MOPS, pH 7.5 to 2mL of 10mg of CREBtide (0.1M MOPS pH 7.5 final concentration).
- Read OD205, OD280 (using 0.1M MOPS buffer as blank)
- Affigel-10 is stored frozen at < -70¼C. Thaw at 4¼C for ~20min.
- Filter 1-2mL on nylon (0.22uM poresize), using Buchner funnel and vacuum. Do not allow beads to dry.
- Wash bed with 3x2mL dH2O.
- Scrape 0.5-1mL into 15mL microfuge tube.
- Add 2.22ml CREBtide ligand solution to tube.
Rock at 4¼C for 4hrs.
- Transfer slurry to column.
- Wash 2x500uL dH2O.
- Collect eluate and save at -20¼C. Read OD.
- Wash column:
- 2x5mL cold PBS
- 1x5mL PBS/0.02% NaN3 (1uL NaN3 in5mL PBS)
- Store at 4¼C upright in beaker in PBS/NaN3 + parafilm.
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