{"id":7423,"date":"2016-04-06T08:03:39","date_gmt":"2016-04-05T23:03:39","guid":{"rendered":"http:\/\/mike.md.tsukuba.ac.jp\/terui\/blog\/?p=7423"},"modified":"2021-12-27T09:38:36","modified_gmt":"2021-12-27T00:38:36","slug":"%e4%b8%8d%e6%80%9d%e8%ad%b0%e3%81%aa%e3%83%97%e3%83%ad%e3%83%88%e3%82%b3%e3%83%ab","status":"publish","type":"post","link":"https:\/\/nbsigh2.com\/?p=7423","title":{"rendered":"\u3081\u3061\u3083\u304f\u3061\u3083\u306a\u30d7\u30ed\u30c8\u30b3\u30eb"},"content":{"rendered":"

\u4f8b\u306eSTAP\u7d30\u80de\u4f5c\u6210\u30d7\u30ed\u30c8\u30b3\u30eb<\/a>\u3060\u304c\uff1b
\n\uff11\uff09ATP\u6fc3\u5ea6\u304c\u3081\u3061\u3083\u304f\u3061\u3083\u3060\u3068\u3044\u3046\u30cd\u30c3\u30c8\u306e\u6279\u5224\u3092\u53d7\u3051\u3066 200 nM \u304c\u00a0200 mM \u306b\u8a02\u6b63\u3055\u308c\u305f\u3002
\nApril 5, 2016 \u306e\u8ffd\u52a0\u60c5\u5831\u3068\u3057\u3066\u300cATP\u306f\u30e2\u30eb\u6fc3\u5ea6\u306e\u7406\u8ad6\u5024\u3067\u306f\u306a\u304f\u3001\u6700\u3082\u826f\u3044\u7d50\u679c\u306e\u3067\u305f\u6e2c\u5b9a\u5024\u3060\u3002ATP\u306e\u91cd\u91cf\u306fpH\u5024\u306e\u5909\u5316\u306b\u57fa\u3065\u3044\u3066\u6c7a\u3081\u305f\u3002\u300d\u306a\u3093\u3066\u308f\u3051\u306e\u308f\u304b\u3089\u306a\u3044\u3053\u3068\u304c\u52a0\u308f\u3063\u3066\u307e\u3059\u3002\u306a\u3093\u3067\u3057\u3087\u306d\u30021 m\u306e\u6c34\u306b110.57 mg\u306eATP\u6eb6\u304b\u3057\u3066\u3069\u3046\u3084\u3063\u3066pH\u3092\u6e2c\u5b9a\u3059\u308b\u3093\u3060\u308d\uff1f\u3053\u3046\u306a\u306e\u304b\u306a\uff1f\u300c1 mL\u306e\u6db2\u4f53\u306epH\u3092\u6e2c\u5b9a\u3057\u3064\u3064ATP\u3092\u52a0\u3048\u3066\u3044\u3063\u3066\u3001\u3042\u308bpH\u5024\u306b\u3059\u308b\u3002\u3067\u305d\u306e\u3068\u304d\u306e\u91cd\u3055\u304c110.57 mg\u3060\u3002\u300d\u3068\u3044\u3046\u3053\u3068\u304b\u306a\uff1f\u3042\u308a\u3048\u306a\u3044\u3060\u308d\u3002\u3067\u304d\u306a\u3044\u3060\u308d\u3002<\/p>\n

10.Carefully the discard the supernatant
\n11.Add 500\u03bcl of HBSS and suspend a cell pellet using a 1000\u03bcl-pipette***
\n12.Take out 6\u03bcl of the cell suspension for cell counting
\n13.Add 6\u03bcl of ATP solution<\/p><\/blockquote>\n

\u3053\u3093\u306a\u6709\u52b9\u6570\u5b57\uff11\u6841\u306e\u5b9f\u9a13\u306b110.57\u3068\u6709\u52b9\u6570\u5b57\uff15\u6841\u306e\u8a66\u85ac\u91cf\u3092\u52a0\u3048\u3066\u610f\u5473\u304c\u3042\u308b\u304b\u3088\u3002\u6eb6\u3051\u308b\u304b\u3069\u3046\u304b\u77e5\u3089\u306a\u3044\u3051\u3069\u300c110 mg \u3092 1 mL \u306e\u6c34\u306b\u6eb6\u304b\u3059\u300d\u7a0b\u5ea6\u306e\u7cbe\u5ea6\u3067\u5341\u5206\u3060\u3002\u771f\u9762\u76ee\u306b 0.01 mg\u306e\u7cbe\u5ea6\u3067\u6e2c\u5b9a\u3057\u3066200 mM\u6eb6\u6db2\u3092\u4f5c\u3089\u306a\u3044\u3068\u3044\u3051\u306a\u3044\u306e\u304b\u3088\u3002\u5b66\u751f\u5b9f\u7fd2\u306e\u96fb\u5b50\u5929\u79e4\u306e\u4f7f\u3044\u65b9\u3058\u3083\u306a\u3044\u3093\u3060\u305c\u3002\u610f\u5473\u306e\u306a\u3044\u3053\u3068\u3084\u308b\u306a\u30fb\u3084\u3089\u305b\u308b\u306a\u3002
\nVacanti \u306e\u30d7\u30ed\u30c8\u30b3\u30eb<\/a>\u3067\u306f200 mM ATP \u6eb6\u6db2\u306epH\u306f3.0 \u7a0b\u5ea6\u3060\u305d\u3046\u3067\u3001\u3053\u308c\u3092HBSS\u306bATP\u6eb6\u6db2\u3092\u6ef4\u4e0b\u3057\u3066pH\u304c5.0 \u306b\u306a\u308b\u3088\u3046\u306b\u3059\u308b\u3002\u305d\u306e\u7d50\u679c\u3001ATP\u6fc3\u5ea6\u306f 3-5 mM \u306b\u306a\u308b\u305d\u3046\u306a\u3002\u3053\u3063\u3061\u306e\u307b\u3046\u304c\u3001STAP\u7d30\u80de\u304c\u3067\u304d\u308b\u304b\u3069\u3046\u304b\u306f\u5225\u306b\u3057\u3066\u3001\u672c\u5f53\u3089\u3057\u3044\u3002
\n\uff12\uff09500 ml<\/strong> of HBSS\u3092\u52a0\u3048\u308b\u3068\u3044\u3046\u3068\u3053\u308d\u304c500 \u03bcl<\/strong> of HBSS\u306b\u8a02\u6b63\u3055\u308c\u305f\u3002\u3053\u308c\u306f\u30cd\u30c3\u30c8\u306e\u6307\u6458\u304c\u306a\u304b\u3063\u305f\u3088\u3046\u3067\u3001\u81ea\u5206\u3067\u6c17\u304c\u3064\u3044\u305f\u3088\u3046\u3060\u3002
\n\uff13\uff09\u5728\u7c73\u30dd\u30b9\u30c9\u30af\u3055\u3093\u306e\u672c\u30d6\u30ed\u30b0\u3067\u306e\u6307\u6458\u3000ATP \u304cAdenosin<\/strong>5\u2019-triphosphate disodium salt hydrate\u3000\u3068adenosine \u306e\u6700\u5f8c\u306be \u304c\u629c\u3051\u3066\u3044\u308b\u3053\u3068\u306b\u306f\u307e\u3060\u6c17\u304c\u3064\u3044\u3066\u3044\u306a\u3044\u3088\u3046\u3060\u3002
\n\uff14\uff09\u7ba1\u7406\u8005\u306e\u6307\u6458\uff1b\u6570\u5b57\u3068\u5358\u4f4d\u306e\u9593\u306f\u30b9\u30da\u30fc\u30b9\u304c\u5fc5\u8981\u3000\u306f\u307e\u3060\u6c17\u304c\u3064\u3044\u3066\u3044\u306a\u3044\u3068\u3044\u3046\u304b\u3001\u8ad6\u6587\u306e\u30b3\u30d4\u30da\u3057\u305f\u3053\u3068\u306f\u3042\u308b\u304c\u3001\u81ea\u3089\u66f8\u3044\u305f\u3053\u3068\u304c\u306a\u3044\u304b\u3089\u77e5\u3089\u306a\u3044\u69d8\u3060\u3002
\n\uff15\uff09\u8a66\u85ac\u306e\u6587\u4e2d\u306b\u3064\u3044\u3066\u306e\u5728\u7c73\u30dd\u30b9\u30c9\u30af\u3055\u3093\u306e\u672c\u30d6\u30ed\u30b0\u3067\u306e\u6307\u6458\uff1b\u300cAliquot in 30\u03bcl increments\u3042\u308b\u3044\u306fAliquot in 10\u03bcl increments\u300d\u3000\u306e\u610f\u5473\u306f\u306a\u3093\u3060\uff1f
\n\u3069\u306e\u304f\u3089\u3044\u5206\u6ce8\u3057\u3066\u4fdd\u5b58\u3059\u308b\u304b\u306f\u3001\u3053\u306e\u3042\u3068\u306e\u624b\u9806\u3067\u3001\u4e00\u5ea6\u306b\u3069\u306e\u304f\u3089\u3044\u306e\u91cf\u3092\u4f7f\u3046\u304b\u3001\u8a66\u85ac\u306e\u4fa1\u683c\u306f\u3001\u4fdd\u5b58\u306b\u8010\u3048\u308b\u304b\u7b49\u306e\u8981\u7d20\u3067\u6c7a\u307e\u308b\u3002ATP\u306f\u305d\u3093\u306a\u306b\u9ad8\u4fa1\u306a\u85ac\u54c1\u3067\u306a\u3044\u3088\u3046\u3060\u3057\u3001\uff11\u57f9\u990a\u76bf\u306b 6\u03bcl \u52a0\u3048\u308b\u3088\u3046\u3067\u3001\u3053\u308c\u3092\u4e00\u56de\u306e\u5b9f\u9a13\u3067\uff15\u30b1\u3064\u304f\u308b\u304b\u3089 30 \u03bcl \u306b\u5206\u6ce8\u4fdd\u5b58\u3059\u308b\u3093\u3060\u308d\u3046\u304b\uff1f6\u03bcl x \uff15\u30b1 \u3067 30 \u03bcL \u306f\u7121\u7406\u3067\u3059\u306a\u30024 \u30b1 \u3060\u306a\u3002\u5206\u6ce8\u3059\u308b\u3053\u3068\u307e\u3067\u30d7\u30ed\u30c8\u30b3\u30eb\u306b\u66f8\u304f\u306e\u304c\u3044\u3044\u306e\u304b\u3001\u66f8\u304f\u306e\u306a\u3089\u3001\u305d\u306e\u7406\u7531\u3082\u52a0\u3048\u3066\u4e01\u5be7\u306b\u8a18\u8f09\u3057\u305f\u307b\u3046\u304c\u89aa\u5207\u3060\u3068\u3044\u3046\u3082\u306e\u3060\u3002\u3069\u306e\u304f\u3089\u3044\u5206\u6ce8\u3057\u3066\u4fdd\u5b58\u3059\u308b\u304b\u306f\u3001\u5b9f\u9a13\u624b\u9806\u3067\u304d\u307e\u308b\u304b\u3089\u3001\u5b9f\u9a13\u8005\u306b\u3088\u3063\u3066\u69d8\u3005\u306b\u306a\u308b\u3002\u3060\u304b\u3089\u3044\u3061\u3044\u3061\u66f8\u304f\u5fc5\u8981\u306f\u306a\u3044\u3002
\n\u3042\u308b\u3044\u306f\u3001\u4ed6\u306e\u4eba\u306bSTAP\u7d30\u80de\u3092\u4f5c\u3063\u3066\u3082\u3089\u3044\u305f\u3044\u3093\u3060\u3060\u308d\u3046\u304b\u3089\u3001\u305d\u3057\u3066\u6587\u7ae0\u306e\u9577\u3055\u306b\u5236\u9650\u304c\u306a\u3044\u3093\u3060\u304b\u3089\u3001\u30df\u30b9\u306e\u306a\u3044\u4e01\u5be7\u306a\u8aac\u660e\u306b\u3059\u3079\u304d\u3060\u3088\u306a\u3002\u672c\u5f53\u306b\u5b9f\u9a13\u3084\u3063\u305f\u30fb\u3067\u304d\u305f\u3093\u3060\u308d\u3046\u304b\uff1f\u82f1\u6587\u8ad6\u6587\u306f\u66f8\u3044\u305f\u3053\u3068\u304c\u306a\u3044\u3088\u3046\u3060\u3002\u82f1\u8a9e\u306b\u3064\u3044\u3066\u306f\u3001\u4e01\u5be7\u306a\u6dfb\u524a\u304c\u3042\u308b\u3088\u3002\u300cSTAP HOPE PAGE\u3001\u5c0f\u4fdd\u65b9\u6674\u5b50\u306e\u82f1\u8a9e\u304c\u6d12\u843d\u306b\u306a\u3089\u306a\u3044<\/a>\u300d\u3060\u3002
\n\u7d30\u80de\u57f9\u990a\u306e\u5b9f\u9a13\u306a\u3093\u304b\u3084\u3063\u305f\u3053\u3068\u306e\u7121\u3044\u30b8\u30b8\u30a4\u306e\u8a00\u3044\u304c\u304b\u308a\u304b\u3082\u306d\u3002<\/p>\n","protected":false},"excerpt":{"rendered":"

\t\t\t\t\t\t<\/p>\n","protected":false},"author":2,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[2,8],"tags":[],"class_list":["post-7423","post","type-post","status-publish","format-standard","hentry","category-education","category-fake"],"_links":{"self":[{"href":"https:\/\/nbsigh2.com\/index.php?rest_route=\/wp\/v2\/posts\/7423","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/nbsigh2.com\/index.php?rest_route=\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/nbsigh2.com\/index.php?rest_route=\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/nbsigh2.com\/index.php?rest_route=\/wp\/v2\/users\/2"}],"replies":[{"embeddable":true,"href":"https:\/\/nbsigh2.com\/index.php?rest_route=%2Fwp%2Fv2%2Fcomments&post=7423"}],"version-history":[{"count":1,"href":"https:\/\/nbsigh2.com\/index.php?rest_route=\/wp\/v2\/posts\/7423\/revisions"}],"predecessor-version":[{"id":21278,"href":"https:\/\/nbsigh2.com\/index.php?rest_route=\/wp\/v2\/posts\/7423\/revisions\/21278"}],"wp:attachment":[{"href":"https:\/\/nbsigh2.com\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=7423"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/nbsigh2.com\/index.php?rest_route=%2Fwp%2Fv2%2Fcategories&post=7423"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/nbsigh2.com\/index.php?rest_route=%2Fwp%2Fv2%2Ftags&post=7423"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}