{"id":1798,"date":"2021-09-16T15:05:51","date_gmt":"2021-09-16T15:05:51","guid":{"rendered":"https:\/\/lab.research.sickkids.ca\/sparc-molecular-analysis\/?page_id=1798"},"modified":"2021-09-16T15:05:51","modified_gmt":"2021-09-16T15:05:51","slug":"ammonium-hydroxide-elution","status":"publish","type":"page","link":"https:\/\/lab.research.sickkids.ca\/sparc-molecular-analysis\/ammonium-hydroxide-elution\/","title":{"rendered":"Ammonium Hydroxide Elution"},"content":{"rendered":"<h1 style=\"text-align: center\"><span style=\"color: #000000\"><strong>PROTOCOL<\/strong><\/span><br \/>\n<span style=\"color: #000000\"><strong>ELUTION FROM BEADS USING AMMONIUM HYDROXIDE<\/strong><\/span><\/h1>\n<p style=\"text-align: left\"><span style=\"color: #000000\"><em>This protocol can be used to elute proteins off of beads prior to Mass Spectrometry Analysis. This is an <\/em><\/span><span style=\"color: #000000\"><em>alternative procedure to on-bead tryptic digestion.<\/em><\/span><\/p>\n<p><span style=\"color: #000000\"><strong>Procedure:<\/strong><\/span><\/p>\n<ol>\n<li><span style=\"color: #000000\">Make fresh elution buffer. <\/span>\n<ul>\n<li><strong><span style=\"color: #000000\">Elution buffer (final) \u2013 (5mL) <\/span><\/strong><\/li>\n<li><span style=\"color: #000000\">9.5M NH4OH<\/span><\/li>\n<li><span style=\"color: #000000\">pH 11.0-12.0 0.5mM EDTA\u00a0<\/span><\/li>\n<\/ul>\n<\/li>\n<li><span style=\"color: #000000\">Wash beads 2X with lysis buffer (same lysis buffer that was used to lyse cells).<\/span><\/li>\n<li><span style=\"color: #000000\">Wash beads 2-3X with the same lysis buffer but without detergents, which helps to remove incompatible detergents from sample prior to mass spectrometry analysis.<\/span><\/li>\n<li><span style=\"color: #000000\">Elute proteins 2-3 times with three bead volumes of elution buffer @ 4\u00b0C with end-over-end agitation for 15 minutes. It is recommended that the elution buffer is prepared fresh. Additionally, monitor the pH of the elution solution before use, pH should equal 11-12 for efficient protein elution. Alternatively, 2% formic acid (HCO2H) can be used for elution however acid elution significantly increased the amount of antibody light and heavy chains in the eluate, and is not as volatile. Alternatively, peptides can be used for elution by competitive inhibition.<\/span><\/li>\n<li><span style=\"color: #000000\">Combine Eluates and freeze on dry ice. Bring samples to SPARC facility for drying and endoproteinase digestion or continue to step 6.<\/span><\/li>\n<li><span style=\"color: #000000\">Dry samples in speed-vacuum. Bring samples to SPARC facility for endoproteinase digestion or continue with in-solution digestion protocol.<\/span><\/li>\n<\/ol>\n<p>&nbsp;<\/p>\n<p><a href=\"https:\/\/lab.research.sickkids.ca\/sparc-molecular-analysis\/wp-content\/uploads\/sites\/61\/2017\/12\/Ammonium-Hydroxide-Eluton.pdf\"><span style=\"color: #307897\"><em><strong>Download and save a PDF copy of the Ammonium Hydroxide Elution Protocol.<\/strong><\/em><\/span><\/a><\/p>\n","protected":false},"excerpt":{"rendered":"<p>PROTOCOL ELUTION FROM BEADS USING AMMONIUM HYDROXIDE This protocol can be used to elute proteins off of beads prior to Mass Spectrometry Analysis. This is an alternative procedure to on-bead tryptic digestion. Procedure: Make fresh elution buffer. Elution buffer (final) \u2013 (5mL) 9.5M NH4OH pH 11.0-12.0 0.5mM EDTA\u00a0 Wash beads 2X with lysis buffer (same&hellip;<\/p>\n","protected":false},"author":205,"featured_media":0,"parent":0,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"footnotes":""},"class_list":["post-1798","page","type-page","status-publish","hentry","description-off"],"yoast_head":"<!-- This site is optimized with the Yoast SEO Premium plugin v27.0 (Yoast SEO v27.0) - https:\/\/yoast.com\/product\/yoast-seo-premium-wordpress\/ -->\n<title>Ammonium Hydroxide Elution - SPARC BioCentre Molecular Analysis<\/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:\/\/lab.research.sickkids.ca\/sparc-molecular-analysis\/ammonium-hydroxide-elution\/\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"Ammonium Hydroxide Elution\" \/>\n<meta property=\"og:description\" content=\"PROTOCOL ELUTION FROM BEADS USING AMMONIUM HYDROXIDE This protocol can be used to elute proteins off of beads prior to Mass Spectrometry Analysis. This is an alternative procedure to on-bead tryptic digestion. Procedure: Make fresh elution buffer. 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