{"id":25495,"date":"2020-06-19T14:28:55","date_gmt":"2020-06-19T06:28:55","guid":{"rendered":"http:\/\/biopioneer.com.tw\/?p=25495"},"modified":"2023-05-10T16:45:57","modified_gmt":"2023-05-10T08:45:57","slug":"%e7%b4%b0%e8%83%9e%e6%a0%b8%e7%b4%b0%e8%83%9e%e8%b3%aa%e5%88%86%e9%9b%a2%e8%a9%a6%e5%8a%91%e7%9b%92-nuclearcytosol-fractionation-kit%e8%b2%a8%e8%99%9fktp3001","status":"publish","type":"post","link":"http:\/\/biopioneer.com.tw\/?p=25495","title":{"rendered":"\u7d30\u80de\u6838\/\u7d30\u80de\u8cea\u5206\u96e2\u8a66\u5291\u76d2 | Nuclear\/Cytosol Fractionation Kit\u8ca8\u865fKTP3001"},"content":{"rendered":"

\u7d30\u80de\u6838\/\u7d30\u80de\u8cea\u5206\u96e2\u8a66\u5291\u76d2 | Nuclear\/Cytosol Fractionation Kit\u8ca8\u865fKTP3001<\/a><\/span><\/strong><\/span><\/p>\n

\u7d30\u80de\u6838\u63d0\u53d6\u7269\u7684\u88fd\u5099\u3002\u6240\u5206\u96e2\u7684\u7d30\u80de\u6838\u548c\u7d30\u80de\u8cea\u86cb\u767d\u8cea\u53ef\u9032\u884c\u8f49\u9304\u6d3b\u6027(transcriptional activity)\u3001RNA \u526a\u63a5 (RNA splicing)\u3001\u96fb\u6cf3\u9077\u79fb\u7387\u8b8a\u52d5\u5206\u6790\uff08Electrophoresis Mobility Shift Assay , EMSA\uff09\u3001\u8db3\u8de1\u5206\u6790(footprinting analysis)\u3001\u5831\u544a\u86cb\u767d\u6aa2\u6e2c (\u00a0reporter assays)\u3001\u8f49\u9304\u5206\u6790(transcription assays)\u3001\u9176\u6d3b\u6027\u6aa2\u6e2c (enzyme activity assays)\u3001 \u7d14\u5316\u8abf\u7bc0\u86cb\u767d(regulatory proteins)\u7684\u8d77\u9ede\u3001\u86cb\u767d\u8cea\u5370\u8de1(Western Blot )\u7814\u7a76\u7b49\u3002<\/p>\n

\"\"\u00a0\"\"<\/p>\n

Fig.1. Western blot of specific proteins fraction using Abbkine ExKine\u2122 Nuclear and Cytoplasmic Protein Extraction Kit. Sample: 293T cells lysate. C: cytoplasmic extraction was analyzed using \u03b1-tubulin antibody (A01080). N: nuclear extraction was analyzed using \u03b1-tubulin antibody (A01080).Fig.2. Western blot of specific proteins fraction using Abbkine ExKine\u2122 Nuclear and Cytoplasmic Protein Extraction Kit. Sample: 293T cells lysate. C: cytoplasmic extraction was analyzed using Histone H3 antibody (A01070). N: nuclear extraction was analyzed using Histone H3 antibody (A01070).<\/p>\n

\u7522\u54c1\u4ecb\u7d39:<\/strong><\/span><\/p>\n

Nuclear\/Cytosol Extraction Kit\u8a66\u5291\u76d2\u5167\u5305\u542b\uff0c\u5206\u96e2\u54fa\u4e73\u52d5\u7269\u7d30\u80de\/\u7d44\u7e54\u4e4b\u7d30\u80de\u6838\u548c\u7d30\u80de\u8cea\u6240\u6709\u8a66\u5291\uff0c\u4e5f\u5305\u62ecProtease Inhibitor Cocktail 100X\u3002\u8a72\u8a66\u5291\u76d2\u61c9\u7528\u539f\u7406\u57fa\u65bc\u7d30\u80de\u5728\u4f4e\u6ef2\u7de9\u885d\u6db2\u4e2d\u6eb6\u8139\u7684\u529f\u80fd\u3002\u7136\u5f8c\u662f\u7d30\u80de\u88ab\u7834\u58de\uff0c\u53bb\u9664\u7d30\u80de\u8cea\u90e8\u5206\uff0c\u91cb\u653e\u6838\u86cb\u767d\u901a\u904e\u9ad8\u9e7d\u7de9\u885d\u6db2\u5f9e\u7d30\u80de\u6838\u4e2d\u5206\u96e2\u51fa\u4f86\u3002\u975e\u8b8a\u6027\u7684\u6d3b\u6027\u86cb\u767d\u53ef\u5728\u77ed\u6642\u9593\u5167\u7d14\u5316\u3002\u5206\u96e2\u7d30\u80de\u6838\u548c\u7d30\u80de\u8cea\u86cb\u767d\u8cea\uff0c\u800c\u4e14\u53ef\u4ee5\u4fdd\u6301\u86cb\u767d\u8cea\u7684\u5b8c\u6574\u6027\u3002 2\u00d7106 <\/sup>cells \u621630~60 mg\u7d44\u7e54\/\u53cd\u61c9\u3002\u53ef\u61c9\u7528\u65bc\u54fa\u4e73\u52d5\u7269\u57f9\u990a\u7d30\u80de\u6216\u7d44\u7e54\u6a23\u54c1\u3002<\/p>\n

\"KTP3001\"<\/p>\n

\u7d30\u80de\u6838\/\u7d30\u80de\u8cea\u5206\u96e2\u8a66\u5291\u76d2KTP3001\u64cd\u4f5c<\/strong><\/span><\/p>\n

    \n
  1. Vortex the tube vigorously on the highest setting for 15 seconds to fully suspend the cell<\/li>\n
  2. Add 10 uL ice-cold CES B to the tube. Vortex the tube for 10-15 seconds on the highest setting. Incubate tube on ice for 1–2 minute.<\/li>\n
  3. Centrifuge the tube at 16000 g for 5 minutes at 4 \u00b0C.<\/li>\n
  4. Immediately transfer the supernatant (cytoplasmic extract) to a clean pre-chilled tube. Place this tube on ice until use or at -80\u00b0C for longer storage. The pellet (which contains nuclei) is usually viscous and not very compact. Optional: In order to remove residual cytoplasmic protein from the nuclei, rinse the pellet with additional ice-cold CES A buffer or PBS. And then repeat procedure C Step 3 and 4.<\/li>\n
  5. Suspend the pellet in 100 uL ice-cold NES. Place the sample on ice and continue vortexing for 15 seconds every 10 minutes, for a total of 30 minutes. Avoid foam formation.<\/li>\n
  6. Centrifuge at 16000 g for 15 minutes at 4\u00b0C.<\/li>\n
  7. Aliquot supernatant (nuclear extract) to clean chilled tubes, keeping a small aliquot aside for protein quantification, flash freeze and store at -80 oC. Avoid freeze\/thaw cycles. Note: The nuclear proteins extracted according to the protocol are suspended in NES, a high salt buffer. If large volumes of nuclear extract are required in subsequent applications or if problems occur with downstream assays, dialyze the nuclear extract to remove excess salts before use.<\/li>\n<\/ol>\n

    \"\u7db2\u7ad9\u898f\u683c\u5c0f\u5716_\u5de5\u4f5c\u5340\u57df<\/a><\/p>\n

    \u6d3b\u7d30\u80de\u548c\u6b7b\u7d30\u80de\u96d9\u67d3\u8272\u5957\u7d44\u00a0<\/a>\u00a0|\u00a0\u00a0CCK-8<\/u><\/strong>\u7d30\u80de\u589e\u6b96\u548c\u7d30\u80de\u6bd2\u6027\u8a66\u5291\u76d2<\/u><\/strong>\u00a0<\/a>\u00a0|\u00a0LDH\u7d30\u80de\u6bd2\u6027\u6e2c\u5b9a\u8a66\u5291\u76d2<\/a>\u00a0|\u00a0\u7d30\u80de\u8870\u8001\u6aa2\u6e2c\u8a66\u5291\u76d2\uff08\u03b2-\u534a\u4e73\u7cd6\u82f7\u9176<\/a>\u00a0|\u00a0\u7d30\u80de\u589e\u6b96EdU Image\u8a66\u5291\u76d2\u00a0<\/a>\u00a0|\u00a0\u7d30\u80de\u6838\/\u7d30\u80de\u8cea\u5206\u96e2\u8a66\u5291\u76d2<\/a><\/span><\/p>\n

    \u80de\u5668\u86cb\u767d\u5206\u96e2\u7684\u61c9\u7528<\/strong><\/span><\/p>\n

      \n
    1. \u63a2\u8a0e\u7d30\u80de\u6838\u5167\u8f49\u9304\u56e0\u5b50\u548c\u7279\u5b9aDNA\u5e8f\u5217\u76f8\u4e92\u4f5c\u7528\u3002\u6aa2\u6e2c\u86cb\u767d\u8cea\u548c\u4e00\u6bb5\u7279\u5b9aDNA\u5e8f\u5217(label)\u76f8\u4e92\u7d50\u5408\u7684\u6280\u8853\u539f\u7406: \u7576\u7279\u5b9a\u5e8f\u5217\u7684DNA\u548c\u67d0\u86cb\u767d\u8cea\u7d50\u5408\u5f8c\uff0c\u6574\u500b\u86cb\u767d\u8cea\u8907\u5408\u9ad4\u5206\u5b50\u91cf\u6703\u589e\u52a0,\u7d93\u7531\u96fb\u6cf3\u5f8c,\u5728gel\u4e2d\u7684\u79fb\u52d5\u901f\u5ea6\u8b8a\u6162,\u6574\u500bband\u9032\u4e0a\u53ef\u52a0\u5165\u7279\u5b9a\u6297\u9ad4\u6aa2\u6e2c\u662f\u5426\u6709\u7279\u5b9a\u86cb\u767d\u8ceaBinding( Supershift)<\/li>\n
    2. \u7d30\u80de\u51cb\u4ea1\u6642,\u7c92\u7dda\u9ad4\u819c\u96fb\u4f4d\u53bb\u6975\u5316\u7684\u904e\u7a0b\u6703\u589e\u52a0\u819c\u7684\u901a\u900f\u6027\u4e26\u4f7f\u819c\u5167\u86cb\u767d,\u5982Cytochrome C, AIF, Smac\/DIABLO\u7b49\u5206\u5b50\u91cb\u653e\u5230\u7d30\u80de\u8cea\u4e2d,\u5f15\u767ccaspase-9\u6d3b\u5316, \u9020\u6210\u4e00\u9023\u4e32\u51cb\u4ea1\u53cd\u61c9\u767c\u751f. COX4\u86cb\u767d\u4f4d\u65bc\u7c92\u817a\u9ad4\u7d30\u80de\u5167\u5074, \u4e26\u5728Apoptosis\u767c\u751f\u6642\u4f9d\u820a\u4fdd\u7559\u5728\u7c92\u7dda\u9ad4\u5167\u3002<\/li>\n
    3. \u4e8c\u7dad\u96fb\u6cf3\u8207MDLC(\u6db2\u76f8\u5c64\u6790)\u4e26\u5217\u70ba\u7814\u7a76\u86cb\u767d\u8cea\u9ad4\u5b78\u7684\u91cd\u8981\u6280\u8853\u4e4b\u4e00\u3002\u539f\u7406: \u5229\u7528\u5206\u5b50\u91cf(\u4e00\u7dad)\u8207\u7b49\u96fb\u9ede(\u4e8c\u7dad)\u4e00\u6b21\u53ef\u5206\u96e2\u5927\u7d042000\u500b\u86cb\u767d\u8cea\u3002\u9650\u5236: \u819c\u86cb\u767d(Hydrophobic)\u5728\u96fb\u6cf3\u4e2d\u4e0d\u6613\u770b\u5230.\u86cb\u767d\u8cea\u82e5\u662f\u70balow abundant protein\u5247\u6b32\u5f97\u5230\u6e96\u78ba\u800c\u53ef\u4fe1\u7684\u5be6\u9a57\u7d50\u679c\u8981\u505a\u80de\u5668\u5206\u96e2( sample prefractionation) \u80de\u5668\u5206\u96e2\u57282d\u61c9\u7528\u7684\u512a\u9ede: \u79fb\u53bbabundant protein\u00a0\u3001\u589e\u52a0 low abundant protein\u3001\u6e1b\u5c11\u86cb\u767d\u8cea\u7684\u8907\u5408\u9ad4\u767c\u751f<\/li>\n<\/ol>\n

       <\/p>\n","protected":false},"excerpt":{"rendered":"

      \u7d30\u80de\u6838\/\u7d30\u80de\u8cea\u5206\u96e2\u8a66\u5291\u76d2 | Nuclear\/Cytosol Fractionation Kit\u8ca8\u865fKTP30 […]<\/p>\n","protected":false},"author":3,"featured_media":24734,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":[],"categories":[3],"tags":[1465],"_links":{"self":[{"href":"http:\/\/biopioneer.com.tw\/index.php?rest_route=\/wp\/v2\/posts\/25495"}],"collection":[{"href":"http:\/\/biopioneer.com.tw\/index.php?rest_route=\/wp\/v2\/posts"}],"about":[{"href":"http:\/\/biopioneer.com.tw\/index.php?rest_route=\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"http:\/\/biopioneer.com.tw\/index.php?rest_route=\/wp\/v2\/users\/3"}],"replies":[{"embeddable":true,"href":"http:\/\/biopioneer.com.tw\/index.php?rest_route=%2Fwp%2Fv2%2Fcomments&post=25495"}],"version-history":[{"count":3,"href":"http:\/\/biopioneer.com.tw\/index.php?rest_route=\/wp\/v2\/posts\/25495\/revisions"}],"predecessor-version":[{"id":42328,"href":"http:\/\/biopioneer.com.tw\/index.php?rest_route=\/wp\/v2\/posts\/25495\/revisions\/42328"}],"wp:featuredmedia":[{"embeddable":true,"href":"http:\/\/biopioneer.com.tw\/index.php?rest_route=\/wp\/v2\/media\/24734"}],"wp:attachment":[{"href":"http:\/\/biopioneer.com.tw\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=25495"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"http:\/\/biopioneer.com.tw\/index.php?rest_route=%2Fwp%2Fv2%2Fcategories&post=25495"},{"taxonomy":"post_tag","embeddable":true,"href":"http:\/\/biopioneer.com.tw\/index.php?rest_route=%2Fwp%2Fv2%2Ftags&post=25495"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}