蛋白纯化操作.doc

His-tag融合蛋白的纯化 The following protocols is for His Bind resin His Bind Purification 1) Express and harvest cells. Resuspend cells in Binding Buffer. Lyse cells.2) Prepare resin (~20- 25 ml?) in column - wet column and frit, transfer resin slurry to column. Wash with water (3 volume), Charge Buffer (5 volumn), Binding Buffer (3 volume).3) Load column with cell lysate, at a rate of ~10 column volumn per hour.4) Wash column (10 X volumn) with Binding Buffer5) Wash column (6 X volumn) with Wash Buffer6) Elute protein (6 X volume) with Elute Buffer (strip buffer can also be used to elute). Alternatively elute first with lower concentration of imidazole at 0.25 M.7) To store column, wash with Strip Buffer (3 X volumn) and stored in Strip Buffer at 4 °C. 1X Buffer reagents Binding Buffer Wash Buffer Elute Buffer Strip Buffer Charge Buffer Tris-HCl pH7.9 20 mM 20 mM 20 mM 20 mM Imidazole 5 mM 60 mM 1M NaCl 0.5 M 0.5 M 0.5 M 0.5 M EDTA 100 mM NiSO4 50 mM Purification in Denaturing Condition (protein in inclusion body)1) Same as before.2) Centrifuge to collect inclusion body. Decant supernatant, resuspend in Binding Buffer (sonicate if necessary), centrifuged. Repeat until all trapped proteins are released.3) Decant supernatant, resuspend in Binding Buffer + 6 M guanidine or Urea. Incubate on ice for 1 hour to dissolve. 4) Centrifuged at 39,000 g for 20 min. Filter supernatant (0.45 micron membrane).5) Load onto column. Purification same as before, except that all buffer contain denaturant, and lower imidazole concentration in Wash Buffer (20 mM) and Elute Buffer (~300mM) (dilute Wash and Elute Buffer with Binding Buffer). Alternatively, you can wash and elute the protein without imidazole at low pH (wash at pH 6.5, elute at pH5.9 or pH4.5 if the protein failed to elute at the hight pH) Regeneration of Column- when flow rate is slow and column resin doesnt turn blue-green when charged1) Wash with 2 vol 6 M guanidine-HCl, then 3 vol