{"id":397,"date":"2018-04-17T18:23:38","date_gmt":"2018-04-17T18:23:38","guid":{"rendered":"https:\/\/wp.uthscsa.edu\/biochemistry\/?page_id=397"},"modified":"2018-04-17T18:23:38","modified_gmt":"2018-04-17T18:23:38","slug":"minimal-biotinylation-cspr","status":"publish","type":"page","link":"https:\/\/lsom.uthscsa.edu\/biochemistry\/minimal-biotinylation-cspr\/","title":{"rendered":"Minimal biotinylation CSPR"},"content":{"rendered":"<p>This protocol is used to minimally biotinylate proteins for capture on a strepavidin or neutravidin sensor chip. Capture of biotinylated proteins provides a more uniform orientation of proteins on the surface than does amine coupling.<\/p>\n<p>You will need:<\/p>\n<ul>\n<li id=\"menus_css_item_15\" class=\"menus_css_el\">EZ-Link\u00ae Sulfo-NHS-LC-LC-Biotin, Pierce, cat no 21338 (<a class=\"menus_css_el\" href=\"http:\/\/cspr.uthscsa.edu\/protocols\/lib\/EZlinkSulfoNHSLCLCBiotin.pdf\">Manufacturer&#8217;s information<\/a>)<\/li>\n<li>Three 50 \u00b5l aliquots of protein to be biotinylated at a concentration between 1 &#8211; 10 mg\/ml. The protein must be in a non-amine containing buffer.<\/li>\n<li>Ice water bath<\/li>\n<li id=\"menus_css_item_16\" class=\"menus_css_el last\">Zeba Desalting Spin Column, Pierce, cat no 89882 (<a class=\"menus_css_el\" href=\"http:\/\/cspr.uthscsa.edu\/protocols\/lib\/ZebaDesaltSpinColumn.pdf\">Manufacturer&#8217;s information<\/a>)<\/li>\n<\/ul>\n<p>Procedure:<\/p>\n<ul>\n<li>Biotinylation is a random event. In order to protect the binding site of some proteins, it may be necessary to biotinylate in the presence of the binding partner and then dissociate.<\/li>\n<li>Weigh out approximately 1 mg of EZ-Link\u00ae Sulfo-NHS-LC-LC-Biotin and add appropriate volume of water for a 1 mg\/ml solution. Use immediately.<\/li>\n<li>To one aliquot of protein, add 10 \u00b5l of EZ-Link\u00ae Sulfo-NHS-LC-LC-Biotin\u00a0<span style=\"color: red\">(A)<\/span>. To the second aliquot, add 2 \u00b5l of EZ-Link\u00ae Sulfo-NHS-LC-LC-Biotin\u00a0<span style=\"color: red\">(B)<\/span>, and to the third aliquot, add 0.4 \u00b5l of EZ-Link\u00ae Sulfo-NHS-LC-LC-Biotin\u00a0<span style=\"color: red\">(C)<\/span>.<\/li>\n<li>Incubate in ice for 30 min. The goal is minimal biotinylation.<\/li>\n<li>While the proteins are incubating, prepare the Zeba desalting columns. The columns contain sodium azide and must be rinsed thoroughly. Add 200 \u00b5l of water or buffer and spin in a \u00b5fuge for 2 minutes at 1,500 x g (4800 rpm in lab microfuge). Repeat four (4) more times discarding the eluate. Keep the column clean and moist until the protein is added.<\/li>\n<li>After incubation, carefully pipet the protein samples into three Zeba desalting columns. Notice that the manufacturer recommends adding 15 \u00b5l of water or buffer to column before adding sample if the volume is less than 70 \u00b5l. Spin in a \u00b5fuge according to the instructions that accompany the Zeba desalting columns. This step is necessary to remove free biotin from the sample.<\/li>\n<li>Recover the protein and store appropriately.<\/li>\n<li>Not knowing the extent of biotinylation, start with capture of tube<span style=\"color: red\">\u00a0(C)<\/span>\u00a0to strepavidin (neutravidin) surface. If the desired capture is not achieved, continue with tube\u00a0<span style=\"color: red\">(B)<\/span>. If still not high enough, finish with tube\u00a0<span style=\"color: red\">(A<\/span><\/li>\n<\/ul>\n","protected":false},"excerpt":{"rendered":"<p>This protocol is used to minimally biotinylate proteins for capture on a strepavidin or neutravidin sensor chip. Capture of biotinylated proteins provides a more uniform orientation of proteins on the surface than does amine coupling. You will need: EZ-Link\u00ae Sulfo-NHS-LC-LC-Biotin, Pierce, cat no 21338 (Manufacturer&#8217;s information) Three 50 \u00b5l aliquots of protein to be biotinylated [&hellip;]<\/p>\n","protected":false},"author":161,"featured_media":0,"parent":0,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"footnotes":""},"class_list":["post-397","page","type-page","status-publish","hentry"],"yoast_head":"<!-- This site is optimized with the Yoast SEO plugin v28.2 - https:\/\/yoast.com\/product\/yoast-seo-wordpress\/ -->\n<title>Minimal biotinylation CSPR - Biochemistry<\/title>\n<meta name=\"robots\" content=\"index, follow, max-snippet:-1, max-image-preview:large, max-video-preview:-1\" \/>\n<link rel=\"canonical\" href=\"https:\/\/lsom.uthscsa.edu\/biochemistry\/minimal-biotinylation-cspr\/\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"Minimal biotinylation CSPR - Biochemistry\" \/>\n<meta property=\"og:description\" content=\"This protocol is used to minimally biotinylate proteins for capture on a strepavidin or neutravidin sensor chip. Capture of biotinylated proteins provides a more uniform orientation of proteins on the surface than does amine coupling. 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Capture of biotinylated proteins provides a more uniform orientation of proteins on the surface than does amine coupling. 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