{"id":2406,"date":"2011-11-16T13:58:24","date_gmt":"2011-11-16T04:58:24","guid":{"rendered":"http:\/\/dna.brc.riken.jp:8002\/?page_id=2406"},"modified":"2018-10-22T09:38:13","modified_gmt":"2018-10-22T00:38:13","slug":"mannual_r","status":"publish","type":"page","link":"http:\/\/dna.brc.riken.jp\/ja\/manual\/mannual_r","title":{"rendered":"\u767a\u73fe\u30d9\u30af\u30bf\u30fc\u306e\u4f7f\u7528"},"content":{"rendered":"

GFP\u767a\u73fe\u30d9\u30af\u30bf\u30fcpCMFlag_EGFP<\/a> (RDB06071)\u306a\u3089\u3073\u306blacZ\u767a\u73fe\u30d9\u30af\u30bf\u30fcpCMFlag_lacZ<\/a> (RDB06072)\u3092Cos-1\u7d30\u80de\u306b\u907a\u4f1d\u5b50\u5c0e\u5165\u3057\u3001\u9855\u5fae\u93e1\u89b3\u5bdf\u306b\u3088\u308bX-gal\u306e\u6d3b\u6027\u67d3\u8272\u3068GFP\u86cd\u5149\u306e\u89b3\u5bdf\u3001\u306a\u3089\u3073\u306b\u30a6\u30a8\u30b9\u30bf\u30f3\u30d6\u30ed\u30c3\u30c8\u306b\u3088\u308bGFP\u3001b-gal\u304a\u3088\u3073FLAG\u306e\u691c\u51fa\u4f8b\u3092\u8a18\u8f09\u3057\u3066\u3044\u307e\u3059\u3002<\/p>\n

\u9855\u5fae\u93e1\u89b3\u5bdf\u306b\u3088\u308bGFP\u306a\u3089\u3073\u306blacZ\u306e\u691c\u51fa<\/h3>\n
    \n
  1. \u3042\u3089\u304b\u3058\u308112-well\u30d7\u30ec\u30fc\u30c8\u306b\u57f9\u990a\u305790%\u30b3\u30f3\u30d5\u30eb\u30a8\u30f3\u30c8\u3068\u306a\u3063\u305fCos-1\u7d30\u80de\u306b\u3001\u907a\u4f1d\u5b50\u5c0e\u5165\u8a66\u85acLipofectamine 2000 (Invitrogen) \u3092\u4f7f\u7528\u3057\u3066\u30d7\u30e9\u30b9\u30df\u30c9\u3092\u5c0e\u5165\u3057\u305f\u3002
    \n

    \u907a\u4f1d\u5b50\u5c0e\u5165\u624b\u9806 (well\u3042\u305f\u308a)
    \nA. Lipofectamine 2000 (Invitrogen), 4 uL plus OptiMEMI (Invitrogen) 500 uL
    \nB. DNA 1.6 ug plus OptiMEMI 500 uL
    \n\u4e0a\u8a18A B \u3092\u6df7\u548c\u5f8c\u3001\u57f9\u990a\u6db2\u3068\u5165\u308c\u63db\u3048\u305f\u3002
    \n6 hr\u5f8c\u3001\u901a\u5e38\u306e\u57f9\u990a\u6db2\u3068\u5165\u308c\u63db\u3048\u305f\u3002<\/blockquote>\n<\/li>\n

  2. \u907a\u4f1d\u5b50\u5c0e\u516524\u6642\u9593\u5f8c\u3001PBS(-)\u30672\u56de\u6d17\u6d44\u3057\u305f\u3002<\/li>\n
  3. 0.25% \u30b0\u30eb\u30bf\u30eb\u30a2\u30eb\u30c7\u30d2\u30c9\u3092500 uL\/well\u6dfb\u52a0\u3057\u305f\u3002<\/li>\n
  4. 4\u2103\u3067over night\u30a4\u30f3\u30ad\u30e5\u30d9\u30fc\u30b7\u30e7\u30f3\u3057\u305f\u3002<\/li>\n
  5. PBS(-)\u30675\u56de\u6d17\u6d44\u3057\u305f\u3002<\/li>\n
  6. X-gal\u67d3\u8272\u6eb6\u6db2\u3092500 uL\/well\u305a\u3064\u6dfb\u52a0\u3057\u305f\u3002
    \n

    X-gal\u67d3\u8272\u6eb6\u6db2
    \n50mM \u30d5\u30a7\u30ea\u30b7\u30a2\u30f3\u5316\u30ab\u30ea\u30a6\u30e0 300 uL
    \n50mM \u30d5\u30a7\u30ed\u30b7\u30ab\u30f3\u5316\u30ab\u30ea\u30a6\u30e0 300 uL
    \n1M MgCl2<\/sub> 0.6 uL
    \n20mg\/mL X-gal 150 uL
    \nPBS(-) 750 uL<\/blockquote>\n<\/li>\n

  7. 37\u2103\u30674hr\u4fdd\u6e29\u3057\u305f\u3002<\/li>\n
  8. PBS(-)\u30672\u56de\u6d17\u6d44\u3057\u305f\u3002<\/li>\n
  9. \u9855\u5fae\u93e1\u660e\u8996\u91ce\u306a\u3089\u3073\u306b\u86cd\u5149\u4e0b\u3067\u89b3\u5bdf\u3057\u305f\u3002<\/li>\n<\/ol>\n

     <\/p>\n\n\n\n\n\n\n
    Detection under microscope<\/th>\n<\/tr>\n
    <\/td>\npCMFlag_EGFP (RDB06071)<\/a><\/td>\npCMFlag_lacZ (RDB06072)<\/a><\/td>\n<\/tr>\n
    fluorescent image<\/td>\n\"\"<\/td>\n\"\"<\/td>\n<\/tr>\n
    X-gal staining<\/td>\n\"\"<\/td>\n\"\"<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

     <\/p>\n

    \u30a6\u30a8\u30b9\u30bf\u30f3\u30d6\u30ed\u30c3\u30c8\u306b\u3088\u308bGFP\u3001beta-galactosidase\u306a\u3089\u3073\u306bFLAG\u30bf\u30b0\u306e\u691c\u51fa<\/h3>\n
      \n
    1. \u3042\u3089\u304b\u3058\u308112-well\u30d7\u30ec\u30fc\u30c8\u306b\u57f9\u990a\u305790%\u30b3\u30f3\u30d5\u30eb\u30a8\u30f3\u30c8\u3068\u306a\u3063\u305fCos-1\u7d30\u80de\u306b\u3001\u907a\u4f1d\u5b50\u5c0e\u5165\u8a66\u85acLipofectamine 2000 (Invitrogen) \u3092\u4f7f\u7528\u3057\u3066\u30d7\u30e9\u30b9\u30df\u30c9\u3092\u5c0e\u5165\u3057\u305f\u3002<\/li>\n

      \u907a\u4f1d\u5b50\u5c0e\u5165\u624b\u9806 (well\u3042\u305f\u308a)
      \nA. Lipofectamine 2000 (Invitrogen), 4 uL plus OptiMEMI (Invitrogen) 500 uL
      \nB. DNA 1.6 ug plus OptiMEMI 500 uL
      \n\u4e0a\u8a18A B \u3092\u6df7\u548c\u5f8c\u3001\u57f9\u990a\u6db2\u3068\u5165\u308c\u63db\u3048\u305f\u3002
      \n6 hr\u5f8c\u3001\u901a\u5e38\u306e\u57f9\u990a\u6db2\u3068\u5165\u308c\u63db\u3048\u305f\u3002<\/blockquote>\n

    2. \u5c0e\u5165\u5f8c24\u6642\u9593\u306b100 uL\u306eRIPA\u30d0\u30c3\u30d5\u30a1\u30fc\u3067\u7d30\u80de\u3092\u56de\u53ce\u3057\u3001\u540c\u91cf\u306e2x Laemmli sample buffer\u3092\u52a0\u3048\u305f\u5f8c\u306b95\u21035\u5206\u52a0\u71b1\u3057\u3001\u86cb\u767d\u8cea\u3092\u8abf\u88fd\u3057\u305f\u3002<\/li>\n
    3. SDS-PAGE (12%\u30a2\u30af\u30ea\u30eb\u30a2\u30df\u30c9)\u306b\u3088\u308a10 uL\u306e\u8abf\u88fd\u86cb\u767d\u8cea\u3092\u6cf3\u52d5\u5f8c\u3001PVDF\u819c (Roche)\u306b\u8ee2\u5199\u3057\u305f\u3002<\/li>\n
    4. \u819c\u30925%\u30b9\u30ad\u30e0\u30df\u30eb\u30af\u3067\u30d6\u30ed\u30c3\u30ad\u30f3\u30b0\u5f8c\u30014\u2103\u3067\u4e00\u6b21\u6297\u4f53\u3068over night\u30a4\u30f3\u30ad\u30e5\u30d9\u30fc\u30b7\u30e7\u30f3\u3057\u305f\u3002<\/li>\n

      \u4e00\u6b21\u6297\u4f53
      \nGFP\u2026 anti GFP (FL), rabbit (1,000 \u500d\u5e0c\u91c8; Santacruz Bio technology, sc-8334)
      \nbeta-gal\u2026 anti beta-gal, rabbit (1,000 \u500d\u5e0c\u91c8; Cappel, 55976)
      \nFLAG\u2026 anti-FLAG M2 Monoclonal Antibody (5,000 \u500d\u5e0c\u91c8; SIGMA, F-3165)<\/blockquote>\n

    5. PBS-T\u306710\u5206x3\u56de\u6d17\u6d44\u3057\u305f\u3002<\/li>\n
    6. \u4e00\u6b21\u6297\u4f53\u53cd\u5fdc\u306e\u819c\u3092\u5ba4\u6e29\u3067\u4e8c\u6b21\u6297\u4f53\u30681\u6642\u9593\u30a4\u30f3\u30ad\u30e5\u30d9\u30fc\u30b7\u30e7\u30f3\u3057\u305f\u3002<\/li>\n

      \u4e8c\u6b21\u6297\u4f53
      \nGFP & beta-gal\u2026 anti rabbit IgG-HRP (NEB)
      \nFLAG\u2026anti mouse IgG (H&L)-HRP (BioRad\uff09<\/blockquote>\n

    7. PBS-T\u30675\u5206x3\u56de\u6d17\u6d44\u3057\u305f\u3002<\/li>\n
    8. ECL\u8a66\u85ac (Pharmacia)\u306a\u3089\u3073\u306bLas3000 (Fuji-Film)\u306b\u3088\u308a\u30b7\u30b0\u30ca\u30eb\u3092\u691c\u51fa\u3057\u305f\u3002<\/li>\n<\/ol>\n

       <\/p>\n\n\n\n\n\n\n
      Detection by Western blotting<\/th>\n<\/tr>\n
      with anti-gfp<\/th>\nwith anti-FLAG<\/th>\nwith anti-galactosidase<\/th>\n<\/tr>\n
      \"\"<\/td>\n\"\"<\/td>\n\"\"<\/td>\n<\/tr>\n
      lanes 1 and 2, pCMFlag_EGFP (RDB06071)<\/a>; lanes 3 and 4, pCMFlag_lacZ (RDB06072)<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

       <\/p>\n

      \u30a6\u30a8\u30b9\u30bf\u30f3\u30d6\u30ed\u30c3\u30c8\u306b\u3088\u308bFLAG\u30bf\u30b0\u306e\u691c\u51fa<\/h3>\n
        \n
      1. \u3042\u3089\u304b\u3058\u308124-well\u30d7\u30ec\u30fc\u30c8\u306b\u57f9\u990a\u305790%\u30b3\u30f3\u30d5\u30eb\u30a8\u30f3\u30c8\u3068\u306a\u3063\u305fCos-1\u7d30\u80de\u306b\u3001\u907a\u4f1d\u5b50\u5c0e\u5165\u8a66\u85acLipofectamine 2000 (Invitrogen) \u3092\u4f7f\u7528\u3057\u3066\u30d7\u30e9\u30b9\u30df\u30c9\u3092\u5c0e\u5165\u3057\u305f\u3002
        \n

        \u907a\u4f1d\u5b50\u5c0e\u5165\u624b\u9806 (well\u3042\u305f\u308a)
        \nA. Lipofectamine 2000 (Invitrogen), 2 uL plus OptiMEMI (Invitrogen) 50 uL
        \nB. DNA 0.8 ug plus OptiMEMI 50 uL
        \n\u4e0a\u8a18A B \u3092\u6df7\u548c\u5f8c\u3001\u57f9\u990a\u6db2\u3068\u5165\u308c\u63db\u3048\u305f\u3002
        \n\u3055\u3089\u306bwell\u3042\u305f\u308a100 uL OptiMEMI \u3092\u52a0\u3048\u305f\u3002
        \n6 hr\u5f8c\u3001\u901a\u5e38\u306e\u57f9\u990a\u6db2\u3068\u5165\u308c\u63db\u3048\u305f\u3002<\/blockquote>\n<\/li>\n

      2. \u5c0e\u5165\u5f8c48\u6642\u9593\u306b50 uL\u306eRIPA\u30d0\u30c3\u30d5\u30a1\u30fc\u3067\u7d30\u80de\u3092\u56de\u53ce\u3057\u3001\u540c\u91cf\u306e2x Laemmli sample buffer\u3092\u52a0\u3048\u305f\u5f8c\u306b95\u21035\u5206\u52a0\u71b1\u3057\u3001\u86cb\u767d\u8cea\u3092\u8abf\u88fd\u3057\u305f\u3002<\/li>\n
      3. SDS-PAGE \u306b\u3088\u308a10 uL\u306e\u8abf\u88fd\u86cb\u767d\u8cea\u3092\u6cf3\u52d5\u5f8c\u3001PVDF\u819c (Roche)\u306b\u8ee2\u5199\u3057\u305f\u3002<\/li>\n
      4. \u819c\u30925%\u30b9\u30ad\u30e0\u30df\u30eb\u30af\u3067\u30d6\u30ed\u30c3\u30ad\u30f3\u30b0\u5f8c\u30014\u2103\u3067\u4e00\u6b21\u6297\u4f53\u3068over night\u30a4\u30f3\u30ad\u30e5\u30d9\u30fc\u30b7\u30e7\u30f3\u3057\u305f\u3002
        \n

        \u4e00\u6b21\u6297\u4f53
        \nFLAG\u2026 anti-FLAG M2 Monoclonal Antibody (5,000 \u500d\u5e0c\u91c8; SIGMA, F-3165)<\/blockquote>\n<\/li>\n

      5. PBS-T\u306710\u5206x3\u56de\u6d17\u6d44\u3057\u305f\u3002<\/li>\n
      6. \u4e00\u6b21\u6297\u4f53\u53cd\u5fdc\u306e\u819c\u3092\u5ba4\u6e29\u3067\u4e8c\u6b21\u6297\u4f53\u30681\u6642\u9593\u30a4\u30f3\u30ad\u30e5\u30d9\u30fc\u30b7\u30e7\u30f3\u3057\u305f\u3002\n<\/li>\n

        \u4e8c\u6b21\u6297\u4f53
        \nFLAG\u2026anti mouse IgG (H&L)-HRP (BioRad\uff09<\/blockquote>\n

      7. PBS-T\u30675\u5206x3\u56de\u6d17\u6d44\u3057\u305f\u3002<\/li>\n
      8. ECL\u8a66\u85ac (Pharmacia)\u306a\u3089\u3073\u306bLas3000 (Fuji-Film)\u306b\u3088\u308a\u30b7\u30b0\u30ca\u30eb\u3092\u691c\u51fa\u3057\u305f\u3002<\/li>\n<\/ol>\n
        \"\"\n<\/div>\n

        (2013.11.03 T.M.)<\/p>\n

        \u57f9\u990a\u7d30\u80de\u306e\u30e9\u30a4\u30bb\u30fc\u30c8\u306e\u8abf\u88fd<\/h3>\n

        \u25a0\u3000\u30a6\u30a8\u30b9\u30bf\u30f3\u30d6\u30ed\u30c3\u30c6\u30a3\u30f3\u30b0\u306a\u3044\u3057\u514d\u75ab\u6c88\u964d\u306a\u3069\u306b\u5229\u7528\u3059\u308b\u30e9\u30a4\u30bb\u30fc\u30c8\u306e\u8abf\u88fd\u65b9\u6cd5\u3067\u3059\u3002\u5fc5\u305a\u6c37\u306e\u4e0a\u3067\u51b7\u3084\u3057\u306a\u304c\u3089\u64cd\u4f5c\u3057\u3066\u304f\u3060\u3055\u3044\u3002
        \n\u25a0\u3000\u30a6\u30a7\u30d6\u4e0a\u306e\u300c\u30e9\u30dc\u30de\u30cb\u30e5\u30a2\u30eb\u300d\u306b\u306f\u3001\u30d0\u30c3\u30d5\u30a1\u30fc\u3092\u4e8c\u7a2e\u985e\u3001Lysis buffer\u3068RIPA buffer\u3092\u793a\u3057\u307e\u3057\u305f\u3002\u514d\u75ab\u6c88\u964d\u306b\u306fSDS\u304c\u5165\u3063\u3066\u3044\u306a\u3044RIPA buffer\u304c\u5408\u3044\u307e\u3059\u3002SDS-PAGE\u306b\u3088\u308b\u30a6\u30a8\u30b9\u30bf\u30f3\u30d6\u30ed\u30c3\u30c6\u30a3\u30f3\u30b0\u306b\u7528\u3044\u308b\u3060\u3051\u3067\u3042\u308c\u3070Lysis buffer\u3092\u4f7f\u3044\u307e\u3059\u3002\u30bf\u30f3\u30d1\u30af\u8cea\u5206\u89e3\u9175\u7d20\u963b\u5bb3\u5264 (protease inhibitor)\u306e\u7a2e\u985e\u306f\u3001\u30e9\u30a4\u30bb\u30fc\u30c8\u3092\u7528\u3044\u308b\u305d\u306e\u5f8c\u306e\u5b9f\u9a13\u306b\u3088\u308a\u9078\u629e\u3057\u3066\u304f\u3060\u3055\u3044\u3002\u307e\u305f\u3001\u8131\u30ea\u30f3\u9178\u5316\u963b\u5bb3\u5264\u3001\u8131SUMO\u5316\u963b\u5bb3\u5264 (de-SUMOylation inhibitor)\u3092\u5b9f\u9a13\u306b\u5fdc\u3058\u3066\u9078\u629e\u3057\u307e\u3059\u3002
        \n\u25a0\u3000\u4ee5\u4e0b\u306b\u624b\u9806\u3092\u304a\u793a\u3057\u3057\u307e\u3059\u304c\u3001\u7528\u3044\u308b\u6a5f\u5668\u3001\u7d30\u80de\u3001\u3042\u308b\u3044\u306f\u7528\u3044\u308b\u8a66\u85ac\u306e\u30ed\u30c3\u30c8\u7b49\u306b\u3088\u3063\u3066\u6761\u4ef6\u304c\u7570\u306a\u308b\u5834\u5408\u304c\u3042\u308a\u307e\u3059\u3002
        \n\u25a0\u3000\u30b5\u30f3\u30d7\u30eb\u306e\u8abf\u88fd
        \n1. \u7d30\u80de\u30926-well\u30d7\u30ec\u30fc\u30c8\u306b\u57f9\u990a\u3059\u308b\u3002
        \n2. \u30d7\u30ec\u30fc\u30c8\u3092\u6c37\u4e0a\u306b\u7f6e\u304d\u51b7\u3084\u3057\u306a\u304c\u3089PBS(-)\u3067\uff12\u56de\u6d17\u3046\u3002
        \n3. PBS(-)\u3092\u9664\u304d\u3001150 ul\u306eLysis buffer\u307e\u305f\u306fRIPA buffer\u3092\u7d30\u80de\u306b\u304b\u3051\u308b\u3002
        \n4. \u6c37\u306e\u4e0a\u3067\u51b7\u3084\u3057\u306a\u304c\u3089\u30d7\u30ec\u30fc\u30c8\u3092\u3088\u304f\u63fa\u3059\u308a\u7d30\u80de\u3092\u3051\u3093\u6fc1\u3059\u308b\u3002
        \n5. \u7d30\u80de\u3068\u30d0\u30c3\u30d5\u30a1\u30fc\u30921.5 ml\u30de\u30a4\u30af\u30ed\u30c1\u30e5\u30fc\u30d6\u306b\u79fb\u3059\u3002
        \n6. \u30de\u30a4\u30af\u30ed\u30c1\u30e5\u30fc\u30d6\u3092\u6c37\u4e0a\u306715\u5206\u9593\u51b7\u3084\u3059\u3002
        \n7. 15,000 rpm\u30014\u2103\u300110\u5206\u306e\u9060\u5fc3\u306b\u3088\u308a\u4e0a\u6e05\u3092\u5206\u96e2\u3059\u308b\u3002
        \n8. \u5206\u96e2\u3057\u305f\u4e0a\u6e05\u3092\u65b0\u3057\u3044\u30c1\u30e5\u30fc\u30d6\u306b\u5206\u53d6\u3057\u3001-80\u2103\u306b\u4fdd\u5b58\u3059\u308b\u3002
        \n\u25a0\u3000\u300c\u3069\u3093\u306a\u5834\u5408\u3053\u308c\u304c\u6700\u9069\u300d\u3068\u3044\u3046\u30d7\u30ed\u30c8\u30b3\u30fc\u30eb\u306f\u3042\u308a\u307e\u305b\u3093\u3002\u57f9\u990a\u7d30\u80de\u30e9\u30a4\u30bb\u30fc\u30c8\u306e\u8abf\u88fd\u306b\u95a2\u3057\u3066\u306f\u30cd\u30c3\u30c8\u4e0a\u3067\u3082\u69d8\u3005\u306a\u30d7\u30ed\u30c8\u30b3\u30fc\u30eb\u3092\u5165\u624b\u3059\u308b\u3053\u3068\u304c\u3067\u304d\u307e\u3059\u3002\u3053\u3053\u306b\u793a\u3057\u305f\u65b9\u6cd5\u3082\u305d\u306e\u4e00\u3064\u3068\u8003\u3048\u3066\u304f\u3060\u3055\u3044\u3002\u7d30\u80de\u306e\u7a2e\u985e\u3084\u91cf\u3001\u57f9\u990a\u306e\u72b6\u614b\u3084\u7528\u3044\u308b\u8a66\u85ac\u306b\u4f9d\u5b58\u3057\u3066\u64cd\u4f5c\u306e\u9069\u5207\u306a\u6761\u4ef6\u306f\u7570\u306a\u308a\u307e\u3059\u306e\u3067\u3001\u4f7f\u7528\u3059\u308b\u8a66\u85ac\u91cf\u3084\u30a4\u30f3\u30ad\u30e5\u30d9\u30fc\u30b7\u30e7\u30f3\u6642\u9593\u3092\u5909\u3048\u3066\u4e88\u5099\u5b9f\u9a13\u3092\u884c\u3044\u3001\u5b9f\u9a13\u3092\u500b\u5225\u306b\u6700\u9069\u5316\u3059\u308b\u5fc5\u8981\u304c\u3042\u308a\u307e\u3059\u3002\u307e\u305f\u3001\u300c\u91cf\u306e\u52b9\u679c\u300d\u3001\u3059\u306a\u308f\u3061\u540c\u3058\u6fc3\u5ea6\u306e\u8a66\u85ac\u3092\u4f7f\u3063\u3066\u3044\u3066\u3082\u53cd\u5fdc\u5bb9\u91cf\u304c\u9055\u3046\u305f\u3081\u306b\u7d50\u679c\u304c\u7570\u306a\u308b\u3053\u3068\u3001\u304c\u3042\u308a\u307e\u3059\u306e\u3067\u3001\u5927\u91cf\u8abf\u88fd\u306b\u306f\u6ce8\u610f\u304c\u5fc5\u8981\u3067\u3059\u3002\u4e88\u5099\u5b9f\u9a13\u306f\u672c\u756a\u3068\u540c\u3058\u53cd\u5fdc\u5bb9\u91cf\u3067\u884c\u3046\u3053\u3068\u304c\u539f\u5247\u3067\u3059\u3002\u4e88\u5099\u5b9f\u9a13\u306e\uff11\uff10\u500d\u91cf\u304c\u5fc5\u8981\u3067\u3042\u308c\u3070\u3001\uff11\uff10\u500d\u5bb9\u91cf\u306e\uff11\u672c\u306e\u53cd\u5fdc\u3092\u884c\u3046\u306e\u3067\u306a\u304f\u3001\u540c\u4e00\u5bb9\u91cf\u306e\uff11\uff10\u672c\u306e\u53cd\u5fdc\u3092\u884c\u3046\u3053\u3068\u3092\u304a\u52e7\u3081\u3057\u307e\u3059\u3002(T.M.)<\/p>\n\n\n\n\n\n\n\n\n
        RIPA buffer<\/span><\/td>\nfinal<\/td>\n40 mL<\/span><\/td>\n<\/tr>\n
        10% NP-40<\/td>\n1%<\/td>\n4.0 mL<\/td>\n<\/tr>\n
        sodium deoxycholate<\/td>\n0.25%<\/td>\n0.1g<\/td>\n<\/tr>\n
        0.25 M EDTA, pH 8.0<\/td>\n1 mM<\/td>\n160 uL<\/td>\n<\/tr>\n
        1 M tris, pH 7.8<\/td>\n50 mM<\/td>\n2.0 mL<\/td>\n<\/tr>\n
        5 M NaCl<\/td>\n150 mM<\/td>\n1.2 mL<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

        Add an appropriate volume of inhibitor.<\/span><\/p>\n

        \n\n\n\n\n\n\n\n\n\n
        Lysis buffer<\/span><\/td>\nfinal<\/td>\n40 mL<\/span><\/td>\n<\/tr>\n
        10% SDS<\/td>\n0.1%<\/td>\n0.4 mL<\/td>\n<\/tr>\n
        10% NP-40<\/td>\n1%<\/td>\n4.0 mL<\/td>\n<\/tr>\n
        sodium deoxycholate<\/td>\n0.5%<\/td>\n0.2g<\/td>\n<\/tr>\n
        0.25 M EDTA, pH 8.0<\/td>\n1 mM<\/td>\n160 uL<\/td>\n<\/tr>\n
        1 M tris, pH 7.8<\/td>\n50 mM<\/td>\n2.0 mL<\/td>\n<\/tr>\n
        5 M NaCl<\/td>\n150 mM<\/td>\n1.2 mL<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

        Add an appropriate volume of inhibitor.<\/span><\/p>\n

        \n\n\n\n\n\n\n
        Protease inhibitor<\/span><\/td>\nfinal<\/td>\n40 mL<\/span><\/td>\n<\/tr>\n
        100 mM PMSF<\/td>\n1 mM<\/td>\n0.4 mL<\/td>\n<\/tr>\n
        1 mg\/mL leupeptin<\/td>\n1 ug\/mL<\/td>\n40 uL<\/td>\n<\/tr>\n
        1 mg\/mL pepstatin A<\/td>\n1 ug\/mL<\/td>\n40uL<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n
        \n\n\n\n\n
        de-SUMOylation inhibitor<\/span><\/td>\nfinal<\/td>\n40 mL<\/span><\/td>\n<\/tr>\n
        1 M NEM in ethanol<\/td>\n1 mM<\/td>\n40 uL<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

        (Mail News 2006.09.21 \u63b2\u8f09\u8a18\u4e8b\u3088\u308a)<\/p>\n

        \n2018.03.04\n","protected":false},"excerpt":{"rendered":"

        GFP\u767a\u73fe\u30d9\u30af\u30bf\u30fcpCMFlag_EGFP (RDB06071)\u306a\u3089\u3073\u306blacZ\u767a\u73fe\u30d9\u30af\u30bf\u30fcpCMFlag_lacZ (RDB06072)\u3092Cos-1\u7d30\u80de\u306b\u907a\u4f1d\u5b50\u5c0e\u5165\u3057\u3001\u9855\u5fae\u93e1\u89b3\u5bdf\u306b\u3088\u308bX-gal\u306e\u6d3b\u6027\u67d3\u8272\u3068GFP\u86cd\u5149\u306e\u89b3\u5bdf\u3001\u306a\u3089\u3073\u306b\u30a6\u30a8\u30b9\u30bf\u30f3\u30d6\u30ed\u30c3\u30c8\u306b\u3088\u308bGFP\u3001b-gal\u304a\u3088\u3073FLAG\u306e\u691c\u51fa\u4f8b\u3092\u8a18\u8f09\u3057\u3066\u3044\u307e\u3059\u3002 \u9855\u5fae\u93e1\u89b3\u5bdf\u306b\u3088\u308bGFP\u306a\u3089\u3073\u306blacZ\u306e\u691c\u51fa \u3042\u3089\u304b\u3058\u308112-well\u30d7\u30ec\u30fc\u30c8\u306b\u57f9 […]<\/p>\n","protected":false},"author":9,"featured_media":0,"parent":296,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"_seopress_titles_title":"","_seopress_titles_desc":"","_seopress_robots_index":"","_seopress_robots_follow":"","_seopress_robots_imageindex":"","_seopress_robots_snippet":"","_seopress_robots_primary_cat":"","_seopress_robots_breadcrumbs":"","_seopress_robots_freeze_modified_date":"","_seopress_robots_custom_modified_date":"","_seopress_robots_canonical":"","_seopress_social_fb_title":"","_seopress_social_fb_desc":"","_seopress_social_fb_img":"","_seopress_social_fb_img_attachment_id":0,"_seopress_social_fb_img_width":0,"_seopress_social_fb_img_height":0,"_seopress_social_twitter_title":"","_seopress_social_twitter_desc":"","_seopress_social_twitter_img":"","_seopress_social_twitter_img_attachment_id":0,"_seopress_social_twitter_img_width":0,"_seopress_social_twitter_img_height":0,"_seopress_redirections_value":"","_seopress_redirections_enabled":"","_seopress_redirections_enabled_regex":"","_seopress_redirections_logged_status":"","_seopress_redirections_param":"","_seopress_redirections_type":0,"_seopress_analysis_target_kw":"","footnotes":"","_wp_rev_ctl_limit":""},"class_list":["post-2406","page","type-page","status-publish","hentry"],"_links":{"self":[{"href":"http:\/\/dna.brc.riken.jp\/ja\/wp-json\/wp\/v2\/pages\/2406","targetHints":{"allow":["GET"]}}],"collection":[{"href":"http:\/\/dna.brc.riken.jp\/ja\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"http:\/\/dna.brc.riken.jp\/ja\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"http:\/\/dna.brc.riken.jp\/ja\/wp-json\/wp\/v2\/users\/9"}],"replies":[{"embeddable":true,"href":"http:\/\/dna.brc.riken.jp\/ja\/wp-json\/wp\/v2\/comments?post=2406"}],"version-history":[{"count":6,"href":"http:\/\/dna.brc.riken.jp\/ja\/wp-json\/wp\/v2\/pages\/2406\/revisions"}],"predecessor-version":[{"id":4480,"href":"http:\/\/dna.brc.riken.jp\/ja\/wp-json\/wp\/v2\/pages\/2406\/revisions\/4480"}],"up":[{"embeddable":true,"href":"http:\/\/dna.brc.riken.jp\/ja\/wp-json\/wp\/v2\/pages\/296"}],"wp:attachment":[{"href":"http:\/\/dna.brc.riken.jp\/ja\/wp-json\/wp\/v2\/media?parent=2406"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}