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        Preparation of Affinity Column

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        Preparation of Affinity Column

        SEK 5/3/95

        1.Add 222.2μl 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 < -70℃.Thaw at 4℃ 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.

        Rock at 4℃ for 4hrs.

        8.Transfer slurry to column.

        9.Wash 2x500μl dH2O.

        10.Collect eluate and save at -20℃.Read OD.

        11.Wash column:

        2x5ml cold PBS

        1x5ml PBS/0.02% NaN3 (1μl NaN3 in5ml PBS)

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

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