Preparation of Affinity Column制备亲和层析柱【UCSF】
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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.
