6L5K&
L. YciF6L5E, YciF6L5K の 精 製
変異体は、ÄKTA System (GE ヘルスケア社)にと Histap HP, HiTrap Q HP,
ResourceQカラム(GEヘルスケア社)で、クロマトグラフィーによる精製を行った。
吸着、溶出バッファーは使用直前に0.22µmのフィルターろ過・脱気を行った（表
L.1 ~ L.8）。吸着バッファーで平衡化したカラムにサンプルを吸着させ、カウンタ
ーイオンのまたは競合試薬の混合濃度を変える事により各タンパク質固有の溶出 位置に分画させ分離した。各フラクションをSDS-PAGEで確認した。
続いて、目的のタンパク質が溶出したフラクションを集め、塩を除くために透 析を行った。透析膜は超純水中で5 min煮沸した後、良くすすいだ物を使った。
透析外液は次の操作に合わせて変更した。透析は4 °C、3時間透析した後、透析 外液を新しくして再び4 °C、O/N 行った。透析液は4 °C、15000 rpm、10分遠心 して沈殿を取り除き、SDS-PAGEで確認した。
また、精製したタンパク質を、BCA Protein Assay Regent Kit (PIERCE社) によ り濃度測定した。
表 L.1. YciF変異体 Histrap HP使用時 吸着バッファー組成 20 mM Tris-Cl pH 8.0
150 mM NaCl
表 L.2. YciF変異体Histrap HP使用時バッファー組成 20 mM Tris-Cl pH 8.0
150 mM NaCl 0,5 M immidazole
表 L.3. YciF変異体 HitrapQ使用時 吸着バッファー組成 20 mM Tris-Cl pH 8.0
表 L.4. YciF変異体 HitrapQ使用時 溶出バッファー組成 20 mM Tris-Cl pH 8.0
1 M KCl
表 L.5. YciF変異体 HitrapQ使用時2回目 吸着バッファー組成 20 mM Tris-Cl pH 8.8
表 L.6. YciF変異体 HitrapQ使用時2回目溶出バッファー組成 20 mM Tris-Cl pH 8.0
1 M KCl
表 L.7. YciF変異体 Resource Q使用時 吸着バッファー組成 20 mM Tris-Cl pH 8.0
表 L.8. YciF変異体 Resource Q使用時 溶出バッファー組成 20 mM Tris-Cl pH 10.0
1 M NaCl
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