{"id":630,"date":"2012-10-24T12:27:06","date_gmt":"2012-10-24T11:27:06","guid":{"rendered":"http:\/\/saber.comule.com\/?p=630"},"modified":"2020-11-12T19:01:07","modified_gmt":"2020-11-12T18:01:07","slug":"dot-blot","status":"publish","type":"post","link":"http:\/\/saber-benmimoun.com\/?p=630","title":{"rendered":"Dot Blot"},"content":{"rendered":"<p><strong>But:<\/strong> D\u00e9tection et quantification de l&#8217;ADN ou de l&#8217;ARN ou des prot\u00e9ines cibles.<\/p>\n<p><strong>Diff\u00e9rence avec Southern Blot Northern Blot :<\/strong> d\u00e9p\u00f4t des \u00e9chantillons directement sur une membrane de nitrocellulose, sans s\u00e9paration pr\u00e9alable.<\/p>\n<p>(+) Le dot est bien sur plus rapide est de nature a etre utiliser en routine dans des aplication medicale ou industrielle.<\/p>\n<p>(-) Il donne un r\u00e9sultat de type pr\u00e9sence absence. Il ne donne pas des informations sur la taille de la mol\u00e9cule d\u00e9tect\u00e9e.<\/p>\n<p>(-) Si deux mol\u00e9cules de taille diff\u00e9rentes sont d\u00e9tect\u00e9es, on aura un seul signal.<\/p>\n<p><strong>Principe:<\/strong><\/p>\n<p>D\u00e9p\u00f4t des \u00e9chantillons sur une membrane sous forme de gouttes.<\/p>\n<p>D\u00e9tection: on utilise deux sondes (ADN ou ARN marqu\u00e9es pour la d\u00e9tection de l&#8217;ADN ou l&#8217;ARN. Des Anticorps pour la d\u00e9tection des prot\u00e9ines.)<\/p>\n<ul>\n<li>sonde 1: contre un g\u00e8ne dont on connait le nombre de copies dans le g\u00e9nome, ou contre un ARN ou prot\u00e9ine dont la quantit\u00e9 de est stable durant les diff\u00e9rentes condition test\u00e9es. Cette sonde servira d&#8217;un t\u00e9moin positif et de t\u00e9moin de charge pour pouvoir comparer les bandes de la sonde 2.<\/li>\n<li>sonde 2 : contre un g\u00e8ne, un ARN ou une prot\u00e9ine d&#8217;int\u00e9r\u00eat.<\/li>\n<\/ul>\n<p>Analyse: A l&#8217;oeil nu, ou par\u00a0par le logiciel ImageJ qui permet de calculer l&#8217;intensit\u00e9 de chaque dot sur la membrane.<\/p>\n<p><a href=\"http:\/\/saber.comule.com\/wp-content\/uploads\/2012\/10\/dotblot.png\"><img loading=\"lazy\" decoding=\"async\" class=\"aligncenter size-thumbnail wp-image-634\" title=\"D\u00e9tection de l'ADN par Dot Blot\" src=\"http:\/\/saber.comule.com\/wp-content\/uploads\/2012\/10\/dotblot-150x150.png\" alt=\"\" width=\"150\" height=\"150\" \/><\/a><\/p>\n<p>&nbsp;<\/p>\n<p><iframe loading=\"lazy\" src=\"http:\/\/www.youtube.com\/embed\/EzEeWMOZr3c\" frameborder=\"0\" width=\"560\" height=\"315\"><\/iframe><br \/>\n[spoiler title =&#8221;Protocole&#8221;]<\/p>\n<p>Reagents<br \/>\nTBS:<br \/>\n20 mM Tris-HCl<br \/>\n150 mM NaCl<br \/>\npH 7.5<br \/>\nTBS-T:<br \/>\n0.05% Tween20 in TBS<br \/>\nBSA\/TBS-T:<br \/>\n0.1% BSA in TBS-T<br \/>\nNitrocellulose membrane (BIO-RAD, Trans-Blot, etc).<br \/>\nProcedure<br \/>\n1. Have nitrocellulose membrane ready, draw grid by pencil to indicate the region you are going to blot (see below).<br \/>\n2. Using narrow-mouth pipette tip, spot 2 \u03bcl of samples onto the nitrocellulose membrane at the center of the grid.<br \/>\nMinimize the area that the solution penetrates (usually 3-4 mm diam.) by applying it slowly.<br \/>\n3. Let the membrane dry.<br \/>\n4. Block non-specific sites by soaking in 5% BSA in TBS-T (0.5-1 hr, RT). Use 10cm Petri Dish for reaction chamber.<br \/>\n5. Incubate with primary antibody (0.1-10 \u03bcg\/ml for purified antibody, 1:1000 to 1:100000 dilution for antisera, 1:100 to<br \/>\n1:10000 for hybridoma supernatant) dissolved in BSA\/TBS-T for 30 min at RT.<br \/>\n6. Wash three times with TBS-T (3 x 5 min).<br \/>\n7. Incubate with secondary antibody conjugated with HRP (for optimum dilution, follow the manufacturer\u2019s<br \/>\nrecommendation) for 30 min at RT.<br \/>\n8. Wash three times with TBS-T (15 min x 1, 5 min x 2), then once with TBS (5 min).<br \/>\n9. Incubate with ECL reagent for 1 min, cover with Saran-wrap (remove excessive solution from the surface), and expose<br \/>\nX-ray film in the dark room. Try several different lengths of exposure.<br \/>\n10. Compare the signal from your unknown sample to that of standard and estimate the concentration.<\/p>\n<p>[\/spoiler]<\/p>\n","protected":false},"excerpt":{"rendered":"<p>But: D\u00e9tection et quantification de l&#8217;ADN ou de l&#8217;ARN ou des prot\u00e9ines cibles. Diff\u00e9rence avec Southern Blot Northern Blot : d\u00e9p\u00f4t des \u00e9chantillons directement sur une membrane de nitrocellulose, sans s\u00e9paration pr\u00e9alable. (+) Le dot est bien sur plus rapide est de nature a etre utiliser en routine dans des aplication medicale ou industrielle. (-)&hellip;<\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":[],"categories":[7],"tags":[],"class_list":["post-630","post","type-post","status-publish","format-standard","hentry","category-biologie","category-7","description-off"],"_links":{"self":[{"href":"http:\/\/saber-benmimoun.com\/index.php?rest_route=\/wp\/v2\/posts\/630","targetHints":{"allow":["GET"]}}],"collection":[{"href":"http:\/\/saber-benmimoun.com\/index.php?rest_route=\/wp\/v2\/posts"}],"about":[{"href":"http:\/\/saber-benmimoun.com\/index.php?rest_route=\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"http:\/\/saber-benmimoun.com\/index.php?rest_route=\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"http:\/\/saber-benmimoun.com\/index.php?rest_route=%2Fwp%2Fv2%2Fcomments&post=630"}],"version-history":[{"count":10,"href":"http:\/\/saber-benmimoun.com\/index.php?rest_route=\/wp\/v2\/posts\/630\/revisions"}],"predecessor-version":[{"id":632,"href":"http:\/\/saber-benmimoun.com\/index.php?rest_route=\/wp\/v2\/posts\/630\/revisions\/632"}],"wp:attachment":[{"href":"http:\/\/saber-benmimoun.com\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=630"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"http:\/\/saber-benmimoun.com\/index.php?rest_route=%2Fwp%2Fv2%2Fcategories&post=630"},{"taxonomy":"post_tag","embeddable":true,"href":"http:\/\/saber-benmimoun.com\/index.php?rest_route=%2Fwp%2Fv2%2Ftags&post=630"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}