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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 <-70C. Thaw at 4C 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 4C for 4hrs.

  9. Transfer slurry to column.
  10. Wash 2x500uL dH2O.
  11. Collect eluate and save at -20C. Read OD.
  12. Wash column:

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