{"id":237,"date":"2024-01-03T02:55:14","date_gmt":"2024-01-02T18:55:14","guid":{"rendered":"http:\/\/39.105.184.3\/?p=237"},"modified":"2025-12-21T01:06:13","modified_gmt":"2025-12-20T17:06:13","slug":"apex2-based-protein-proximal-labeling","status":"publish","type":"post","link":"https:\/\/fanyiming.life\/?p=237","title":{"rendered":"APEX2-based Protein Proximal Labeling"},"content":{"rendered":"\n<p>APEX2 Proximal Labeling Protocol<\/p>\n\n\n\n<h1 class=\"wp-block-heading\">Advantage<\/h1>\n\n\n\n<ol class=\"wp-block-list\">\n<li>Capture weak or transient interactive proteins.<\/li>\n\n\n\n<li>Detect proteome at &#8220;inaccessible&#8221; regions of interest which can not be purified, such as mitochondrial intermembrane space (IMS).<\/li>\n<\/ol>\n\n\n\n<h1 class=\"wp-block-heading\">Plasmid<\/h1>\n\n\n\n<p>APEX2-V5-POI<\/p>\n\n\n\n<blockquote class=\"wp-block-quote is-layout-flow wp-block-quote-is-layout-flow\">\n<p>POI: proteins of interest<\/p>\n<\/blockquote>\n\n\n\n<h1 class=\"wp-block-heading\">Buffer<\/h1>\n\n\n\n<ol class=\"wp-block-list\">\n<li>500mM Biotin-phenol\u6bcd\u6db2<\/li>\n<\/ol>\n\n\n\n<p>\u79f0\u53d60.05g Biotin-phenol\uff0c\u6eb6\u4e8e278\u03bcL DSMO\uff0c\u5206\u88c5\uff08\u6bcf\u7ba130\u03bcL\uff09\u540e-20\u2103\u50a8\u5b58.<\/p>\n\n\n\n<figure class=\"wp-block-image size-large\"><img loading=\"lazy\" decoding=\"async\" width=\"1024\" height=\"455\" src=\"https:\/\/fanym.oss-cn-beijing.aliyuncs.com\/wp-content\/uploads\/2024\/05\/20240502131030574-1024x455.png?x-oss-process=style\/default\" alt=\"\" class=\"wp-image-3337\" srcset=\"https:\/\/fanym.oss-cn-beijing.aliyuncs.com\/wp-content\/uploads\/2024\/05\/20240502131030574-1024x455.png?x-oss-process=style\/default 1024w, https:\/\/fanym.oss-cn-beijing.aliyuncs.com\/wp-content\/uploads\/2024\/05\/20240502131030574-300x133.png?x-oss-process=style\/default 300w, https:\/\/fanym.oss-cn-beijing.aliyuncs.com\/wp-content\/uploads\/2024\/05\/20240502131030574-768x341.png?x-oss-process=style\/default 768w, https:\/\/fanym.oss-cn-beijing.aliyuncs.com\/wp-content\/uploads\/2024\/05\/20240502131030574-1536x682.png?x-oss-process=style\/default 1536w, https:\/\/fanym.oss-cn-beijing.aliyuncs.com\/wp-content\/uploads\/2024\/05\/20240502131030574.png?x-oss-process=style\/default 1599w\" sizes=\"auto, (max-width: 1024px) 100vw, 1024px\" \/><\/figure>\n\n\n\n<ol class=\"wp-block-list\" start=\"2\">\n<li>500\u03bcM Biotin-phenol in \u65b0\u9c9c\u57f9\u517b\u57fa 1:1000\u5728\u65b0\u9c9c\u57f9\u517b\u57fa\u4e2d\u52a0\u5165500mM Biotin-phenol\u6bcd\u6db2.<\/li>\n\n\n\n<li>5mM H2O2 in \u65b0\u9c9c\u57f9\u517b\u57fa\uff08\u7b2c3\u6b65\u5feb\u7ed3\u675f\u65f6\u73b0\u914d\uff09 5.11\u03bcL*30%\u8fc7\u6c27\u5316\u6c22\uff0c\u52a0\u516510 mL\u65b0\u9c9c\u57f9\u517b\u57fa\u4e2d.<\/li>\n\n\n\n<li>\u53cd\u5e94\u7ec8\u6b62\u6db2(PBS with 10mM sodium ascorbate, 5mM TROLOX, 10mM sodium azide) \u5148\u914d\u7f6e\u50a8\u6db20.198g sodium ascorbate in 1mL water (100X), 0.125g TROLOX to 1mL DMSO.<\/li>\n<\/ol>\n\n\n\n<blockquote class=\"wp-block-quote is-layout-flow wp-block-quote-is-layout-flow\">\n<p>\u6ce8\u610f\u8fd9\u4e24\u4e2a\u50a8\u6db2\u4e5f\u4e0d\u80fd\u771f\u7684\u53bb\u201c\u50a8\u201d\uff0c\u4f7f\u7528\u65f6\u73b0\u914d\uff01<\/p>\n<\/blockquote>\n\n\n\n<p>\u4f7f\u7528\u65f6\u63091:100\u52a0\u5165100mL PBS\u4e2d.<br>\u200b<br><\/p>\n\n\n\n<figure class=\"wp-block-image size-full\"><img loading=\"lazy\" decoding=\"async\" width=\"800\" height=\"378\" src=\"https:\/\/fanym.oss-cn-beijing.aliyuncs.com\/wp-content\/uploads\/2024\/05\/20240502130959702.png?x-oss-process=style\/default\" alt=\"\" class=\"wp-image-3335\" srcset=\"https:\/\/fanym.oss-cn-beijing.aliyuncs.com\/wp-content\/uploads\/2024\/05\/20240502130959702.png?x-oss-process=style\/default 800w, https:\/\/fanym.oss-cn-beijing.aliyuncs.com\/wp-content\/uploads\/2024\/05\/20240502130959702-300x142.png?x-oss-process=style\/default 300w, https:\/\/fanym.oss-cn-beijing.aliyuncs.com\/wp-content\/uploads\/2024\/05\/20240502130959702-768x363.png?x-oss-process=style\/default 768w\" sizes=\"auto, (max-width: 800px) 100vw, 800px\" \/><\/figure>\n\n\n\n<ol class=\"wp-block-list\" start=\"5\">\n<li>RIPA buffer (\u73b0\u52a0cocktail\u548cPMSF) 50 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, \u4f7f\u7528\u65f6\u73b0\u52a0cocktail\u548cPMSF)\u6709\u7684Protocol\u63a8\u8350\uff1aRIPA lysis buffer supplemented with 1\u00d7 protease inhibitor cocktail, 1 mM PMSF and quenchers (10 mM sodium azide, 10 mM sodium ascorbate and 5 mM Trolox)<\/li>\n\n\n\n<li>1M KCl\u6eb6\u6db2<\/li>\n\n\n\n<li>0.1M Na2CO3\u6eb6\u6db2<\/li>\n\n\n\n<li>2M\u5c3f\u7d20 in 10mM Tris-HCl, pH8.0<\/li>\n\n\n\n<li>\u6d17\u8131\u6db2(3x protein loading buffer supplemented with 20mM DTT and 2mM biotin)                          <br><img loading=\"lazy\" decoding=\"async\" decoding=\"async\" src=\"https:\/\/eamonimgbed.oss-cn-beijing.aliyuncs.com\/img\/202401031937253.png?x-oss-process=style\/screenshots\" alt=\"image-20240103193710105\"><\/li>\n<\/ol>\n\n\n\n<h1 class=\"wp-block-heading\">Procedure<\/h1>\n\n\n\n<h2 class=\"wp-block-heading\">Biotin labeling<\/h2>\n\n\n\n<ol class=\"wp-block-list\">\n<li>\u7ec6\u80de\u4f20\u81f32\u4e2a10cm\u677f\u957f\u6ee1<\/li>\n\n\n\n<li>\u5f03\u57f9\u517b\u57fa\uff0c\u6bcf\u4e2a10cm\u677f\u52a0\u51654mL (500\u03bcM Biotin-phenol in \u65b0\u9c9c\u57f9\u517b\u57fa)<\/li>\n\n\n\n<li>\u653e\u516537\u2103 CO2\u57f9\u517b\u7bb1\u9759\u7f6e30min<\/li>\n\n\n\n<li>\u73b0\u914d1mL (5mM H2O2 in \u65b0\u9c9c\u57f9\u517b\u57fa)\uff0c\u7acb\u523b\u4ece\u57f9\u517b\u7bb1\u4e2d\u53d6\u51fa\u7ec6\u80de\uff0c\u8fc5\u901f\u5c061mL (5mM H2O2 in \u65b0\u9c9c\u57f9\u517b\u57fa)\u52a0\u516510cm\u677f\u4e2d(H2O2\u7ec8\u6d53\u5ea61mM)\uff0c\u5728\u624b\u4e2d\u6e29\u67d4\u6447\u6643\u5b75\u80b21min.\u4e0d\u8981\u540c\u65f6\u64cd\u4f5c\u8d85\u8fc72\u76d8\u7ec6\u80de\u3002<\/li>\n\n\n\n<li>\u8fc5\u901f\u5f03\u4e0a\u6e05\uff0c\u8fc5\u901f\u52a0\u51654mL\u53cd\u5e94\u7ec8\u6b62\u6db2(PBS with 10mM sodium ascorbate, 5mM TROLOX).<\/li>\n\n\n\n<li>\u6e29\u67d4\u6447\u66435s\uff0c\u5f03\u4e0a\u6e05\uff0c\u52a0\u51654mL\u53cd\u5e94\u7ec8\u6b62\u6db2.<\/li>\n\n\n\n<li>\u6e29\u67d4\u6447\u66435s\uff0c\u5f03\u4e0a\u6e05\uff0c\u52a0\u51654mL\u53cd\u5e94\u7ec8\u6b62\u6db2.<\/li>\n\n\n\n<li>\u6e29\u67d4\u6447\u66435s\uff0c\u5f03\u4e0a\u6e05\uff0c\u52a0\u51652mL\u9884\u51b7\u7684PBS.<\/li>\n\n\n\n<li>\u5439\u6253\u7ec6\u80de\uff0c\u6536\u96c6\u81f315mL\u79bb\u5fc3\u7ba1\uff08\u4e24\u4e2a\u677f2mL+2mL\uff09.<\/li>\n\n\n\n<li>3000 x g, 4\u2103, \u79bb\u5fc35cm\uff0c\u5f03\u4e0a\u6e05\uff0c\u7ec6\u80depellet\u51bb\u5b58-80\u2103.<\/li>\n<\/ol>\n\n\n\n<h2 class=\"wp-block-heading\">Streptavidin beads enrichment of biotinylated proteins<\/h2>\n\n\n\n<ol class=\"wp-block-list\">\n<li>\u51b0\u4e0a\u89e3\u51bb\u7ec6\u80de.<\/li>\n\n\n\n<li>\u52a0\u51651.5mL RIPA buffer\uff0c\u5439\u6253\u6df7\u5300\uff0c4\u2103\u6162\u901f\u65cb\u8f6c\u88c2\u89e35min.<\/li>\n\n\n\n<li>15,000 x g, 4\u2103\u79bb\u5fc310min\uff0c\u6536\u96c6\u4e0a\u6e05\u6db2\uff0c\u6d4b\u86cb\u767d\u6d53\u5ea6.<\/li>\n\n\n\n<li>\u53d6450 \u03bcL Streptavidin-coated magnetic beads (Pierce)\uff0cRIPA Buffer\u6d17\u4e24\u6b21.<\/li>\n\n\n\n<li>\u5f03\u4e0a\u6e05\uff0c\u5411beads\u6c89\u6dc0\u4e2d\u52a0\u51658mg\u86cb\u767d\u6eb6\u6db2\uff0c\u5ba4\u6e29\u6162\u901f\u65cb\u8f6c\u5b75\u80b21h\u62164\u2103\u6162\u901f\u65cb\u8f6c\u5b75\u80b2\u8fc7\u591c.<\/li>\n\n\n\n<li>1mL RIPA buffer\u6d17\u4e24\u6b21.<\/li>\n\n\n\n<li>1mL 1M KCl\u6eb6\u6db2\u6d17\u4e00\u6b21.<\/li>\n\n\n\n<li>1mL 0.1M Na2CO3\u6eb6\u6db2\u6d17\u4e00\u6b21.<\/li>\n\n\n\n<li>1mL (2M\u5c3f\u7d20 in 10mM Tris-HCl, pH8.0)\u6d17\u4e00\u6b21.<\/li>\n\n\n\n<li>1mL RIPA Buffer\u6d17\u4e24\u6b21.<\/li>\n\n\n\n<li>\u52a0\u516575\u03bcL (3x protein loading buffer supplemented with 20mM DTT and 2mM biotin)\uff0c\u8dd1SDS-PAGE\u548cStrep-WB.<\/li>\n\n\n\n<li>\u8d28\u8c31\u5206\u6790.<\/li>\n<\/ol>\n\n\n\n<figure class=\"wp-block-image\"><img loading=\"lazy\" decoding=\"async\" decoding=\"async\" src=\"https:\/\/eamonimgbed.oss-cn-beijing.aliyuncs.com\/img\/202401031939010.png?x-oss-process=style\/screenshots\" alt=\"image-20240103193940564\" \/><\/figure>\n\n\n\n<h1 class=\"wp-block-heading\">Optimization<\/h1>\n\n\n\n<h2 class=\"wp-block-heading\">Control 1 \u2014 Negative control [no-H2O2 or no-BP (biotin-phenol)]<\/h2>\n\n\n\n<figure class=\"wp-block-image\"><img loading=\"lazy\" decoding=\"async\" decoding=\"async\" src=\"https:\/\/eamonimgbed.oss-cn-beijing.aliyuncs.com\/img\/202401031947018.png?x-oss-process=style\/screenshots\" alt=\"image-20240103194706550\" \/><\/figure>\n\n\n\n<h2 class=\"wp-block-heading\">Control 2 \u2014 Mass spectrometry control<\/h2>\n\n\n\n<p>\u6839\u636e\u5177\u4f53\u7684\u76ee\u6807\u86cb\u767d\u5b9a\u4f4d\u6765\u9009\u62e9\uff0c\u5355\u7eaf\u7684no-APEX2 or no-H2O2 Control\u6216\u8005\u522b\u7684\u86cb\u767dconstruct\u7684Control<\/p>\n\n\n\n<h2 class=\"wp-block-heading\">Imaging APEX2-V5-POI localization<\/h2>\n\n\n\n<h2 class=\"wp-block-heading\">Optimize Streptavidin pull-down<\/h2>\n\n\n\n<p>\u539f\u6587\u63a8\u8350\uff1a360 \u00b5g (~90 \u00b5l) of each whole-cell lysate sample with 30 \u00b5l of streptavidin magnetic beads (Pierce).<\/p>\n\n\n\n<p>\u53ef\u4ee5\u4f7f\u7528\u5c0f\u6837\uff086-well plate\u7684\u4e00\u5b54\u7ec6\u80de\uff0c\u5927\u69822.5Million\uff09\u8fdb\u884c\u6761\u4ef6\u6478\u7d22\u3002<\/p>\n\n\n\n<h1 class=\"wp-block-heading\">Critical points<\/h1>\n\n\n\n<ol class=\"wp-block-list\">\n<li>\u4e0d\u80fd\u4f7f\u7528BCA\u6cd5\u5bf9\u86cb\u767d\u8fdb\u884c\u5b9a\u91cf\uff0cQuench buffer\u4e2d\u7684\u6210\u5206\u5bf9BCA\u6cd5\u6709\u5e72\u6270\uff1b<\/li>\n\n\n\n<li>\u4e0d\u8981\u5728fusion construct\u4e2d\u4f7f\u7528HA\u6807\u7b7e\uff0cHA\u6807\u7b7e\u53ef\u80fd\u56e0\u4e3a\u88ab\u6807\u8bb0biotin\u800c\u5931\u6548\uff1b<\/li>\n\n\n\n<li>\u4e0d\u8981\u540c\u65f6\u64cd\u4f5c2\u76d8\u4ee5\u4e0a\u7ec6\u80de\uff0c\u4ee5\u514d\u5f71\u54cdH2O2\u6807\u8bb0\u65f6\u95f4\uff0c\u65e0\u6cd5\u53ca\u65f6\u7ec8\u6b62\uff1b<\/li>\n\n\n\n<li>\u6297\u574f\u8840\u9178\u94a0\u548cTROLOX\u4e00\u5b9a\u8981\u4ece\u5e72\u7c89\u73b0\u914d\u73b0\u7528\uff0c\u4e0d\u80fd\u5b58\uff1b<\/li>\n\n\n\n<li>H2O2\u8981\u5728\u5f00\u59cb\u4f7f\u7528\u524d\u51e0\u5206\u949f\u73b0\u914d\uff0c\u4ee5\u514d\u5206\u89e3\uff1b<\/li>\n<\/ol>\n","protected":false},"excerpt":{"rendered":"<p>\u8fd9\u7bc7\u7b14\u8bb0\u8be6\u7ec6\u8bb0\u5f55\u4e86\u57fa\u4e8eAPEX2\u7684\u86cb\u767d\u8d28\u90bb\u8fd1\u6807\u8bb0\u6280\u672f\uff0c\u91cd\u70b9\u9610\u8ff0\u4e86\u5176\u80fd\u6355\u83b7\u5f31\/\u77ac\u65f6\u76f8\u4e92\u4f5c\u7528\u3001\u63a2\u6d4b\u96be\u7eaf\u5316\u533a\u57df\uff08\u5982\u7ebf\u7c92\u4f53\u819c\u95f4\u9699\uff09\u7684\u4f18\u52bf\uff0c\u5e76\u63d0\u4f9b\u4e86\u4ece\u8d28\u7c92\u6784\u5efa\u3001\u5173\u952e\u7f13\u51b2\u6db2\u914d\u5236\u5230\u751f\u7269\u7d20\u6807\u8bb0\u3001\u94fe\u9709\u4eb2\u548c\u7d20\u73e0\u5bcc\u96c6\u53ca\u8d28\u8c31\u5206\u6790\u7684\u5b8c\u6574\u5b9e\u9a8c\u6d41\u7a0b\u4e0e\u4f18\u5316\u8981\u70b9\u3002<\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[4],"tags":[],"class_list":["post-237","post","type-post","status-publish","format-standard","hentry","category-note"],"_links":{"self":[{"href":"https:\/\/fanyiming.life\/index.php?rest_route=\/wp\/v2\/posts\/237","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/fanyiming.life\/index.php?rest_route=\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/fanyiming.life\/index.php?rest_route=\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/fanyiming.life\/index.php?rest_route=\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/fanyiming.life\/index.php?rest_route=%2Fwp%2Fv2%2Fcomments&post=237"}],"version-history":[{"count":2,"href":"https:\/\/fanyiming.life\/index.php?rest_route=\/wp\/v2\/posts\/237\/revisions"}],"predecessor-version":[{"id":14144,"href":"https:\/\/fanyiming.life\/index.php?rest_route=\/wp\/v2\/posts\/237\/revisions\/14144"}],"wp:attachment":[{"href":"https:\/\/fanyiming.life\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=237"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/fanyiming.life\/index.php?rest_route=%2Fwp%2Fv2%2Fcategories&post=237"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/fanyiming.life\/index.php?rest_route=%2Fwp%2Fv2%2Ftags&post=237"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}