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Protocals: Preparation of Affinity Column

Preparation of Affinity Column

SEK 5/3/95
  1. Add 222.2ul 1M MOPS, pH 7.5 to 2mL of 10mg of CREBtide (0.1M MOPS pH 7.5 final concentration).

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

  3. Affigel-10 is stored frozen at <-70¼C. Thaw at 4¼C for ~20min.

  4. Filter 1-2mL on nylon (0.22uM poresize), using Buchner funnel and vacuum. Do not allow beads to dry.

  5. Wash bed with 3x2mL dH2O.

  6. Scrape 0.5-1mL into 15mL microfuge tube.

  7. Add 2.22ml CREBtide ligand solution to tube.

    8. Rock at 4¼C for 4hrs.

  8. Transfer slurry to column.

  9. Wash 2x500uL dH2O.

  10. Collect eluate and save at -20¼C. Read OD.

  11. Wash column:

  12. Store at 4¼C upright in beaker in PBS/NaN3 + parafilm.