{"id":1811,"date":"2021-09-16T15:25:08","date_gmt":"2021-09-16T15:25:08","guid":{"rendered":"https:\/\/lab.research.sickkids.ca\/sparc-molecular-analysis\/?page_id=1811"},"modified":"2021-09-16T15:25:08","modified_gmt":"2021-09-16T15:25:08","slug":"in-gel-digestion","status":"publish","type":"page","link":"https:\/\/lab.research.sickkids.ca\/sparc-molecular-analysis\/in-gel-digestion\/","title":{"rendered":"In-Gel Digestion"},"content":{"rendered":"<h1 style=\"text-align: center\"><span style=\"color: #000000\">IN-GEL DIGESTION AND EXTRACTION PROTOCOL<\/span><\/h1>\n<h4><span style=\"color: #000000\"><strong>Solutions Required: <\/strong><\/span><\/h4>\n<ul>\n<li><span style=\"color: #000000\">1% Acetic Acid<\/span><\/li>\n<li><span style=\"color: #000000\">100% Acetonitrile (ACN), HPLC Grade<\/span><\/li>\n<li><span style=\"color: #000000\">10mM Dithiothreitol (DTT)<\/span><\/li>\n<li><span style=\"color: #000000\">55mM iodoacetamide (IAA), prepared immediately prior to use, protect from light<\/span><\/li>\n<li><span style=\"color: #000000\">25mM NH4HCO3<\/span><\/li>\n<li><span style=\"color: #000000\">Gel Wash Solution<\/span>\n<ul>\n<li><span style=\"color: #000000\">25mM NH4HCO3<\/span><\/li>\n<li><span style=\"color: #000000\">50% ACN<\/span><\/li>\n<\/ul>\n<\/li>\n<li><span style=\"color: #000000\">Enzyme of interest<\/span><\/li>\n<\/ul>\n<h4><span style=\"color: #000000\"><strong>Protocol: <\/strong><\/span><\/h4>\n<p><span style=\"color: #000000\"><strong>Part A: Excision &amp; De-stain of gel bands <\/strong><\/span><\/p>\n<p style=\"padding-left: 40px\"><span style=\"color: #000000\"><strong>A1.<\/strong> Excise protein bands of interest from de-stained gel with sharp scalpel. Ensure to cut as close to the band of interest on all 4 sides to reduce the amount of gel. Optimal results are achieved when minimal gel is present. Place one single band in a microfuge tube for processing. Do not place more than one band in a tube.<\/span><\/p>\n<p style=\"padding-left: 40px\"><span style=\"color: #000000\"><strong>A2<\/strong>. De-stain gel band further in microfuge with vigorous shaking using appropriate de-stain<\/span><br \/>\n<span style=\"color: #000000\">solution for 30 minutes.<\/span><br \/>\n<span style=\"color: #000000\"><strong>A3.<\/strong> Remove de-stain solution by gentle aspiration.<\/span><br \/>\n<span style=\"color: #000000\"><strong>A4.<\/strong> If gel band is still blue, replace solution with fresh de-stain solution and repeat steps A2 &amp;\u00a0A3 until stain is no longer observed.<\/span><br \/>\n<span style=\"color: #000000\"><strong>A5.<\/strong> Cover gel band with 1% acetic acid. At this point, bands in acetic acid may be stored indefinitely at 4oC or they may be processed immediately. Do not freeze gel bands, as this will reduce yield.<\/span><\/p>\n<p><span style=\"color: #000000\"><strong>Part B: Reducing Disulfide Bonds &amp; Alkylation of free cysteine residues <\/strong><\/span><\/p>\n<p style=\"padding-left: 40px\"><span style=\"color: #000000\"><strong>B1.<\/strong> Dehydrate gel pieces with 500\uf06dL of 100% ACN for 10 minutes<\/span><\/p>\n<p style=\"padding-left: 40px\"><span style=\"color: #000000\"><strong>B2.<\/strong> Remove acetonitrile and dry gel pieces in vacuum centrifuge for 10 minutes.<\/span><\/p>\n<p style=\"padding-left: 40px\"><span style=\"color: #000000\"><strong>B3.<\/strong> Add 100uL of 10mM DTT to the gel piece, and incubate for 45 minutes at 55oC.<\/span><\/p>\n<p style=\"padding-left: 80px\"><span style=\"color: #000000\"><em>Dithiothreitol (DTT) is a reducing agent that converts the cysteine\u2019s disulfide bond into free sulfhydryl groups.<\/em><\/span><\/p>\n<p style=\"padding-left: 40px\"><span style=\"color: #000000\"><strong>B4.<\/strong> Remove DTT solution by gentle aspiration and add 100\uf06dL of freshly prepared 55mM iodoacetamide (IAA), and incubate for 20 minutes at room temperature in the dark.<\/span><\/p>\n<p style=\"padding-left: 80px\"><span style=\"color: #000000\"><em>Iodoacetamide (IAA) is an alkylating reagent that reacts with the free sulfhydryl groups of cysteine residues. This reaction forms S-carboxyamidomethyl-cysteine, which cannot be re-oxidized to form disulfide bonds. This step is important to allow the digesting enzyme maximum access to cleavage sites within the protein. If this step is not done, peptides containing cysteine residues will not be identified in the same capacity as if this step is done.\u00a0<\/em><\/span><\/p>\n<p style=\"padding-left: 40px\"><span style=\"color: #000000\"><strong>B5.<\/strong> Remove IAA solution by gentle aspiration<\/span><br \/>\n<span style=\"color: #000000\"><strong>B6.<\/strong> Add 500uL of Gel Wash Solution, and incubate at room temperature with gentle shaking<\/span><br \/>\n<span style=\"color: #000000\">for 15 minutes.<\/span><br \/>\n<span style=\"color: #000000\"><strong>B7.<\/strong> Repeat step B6 twice.<\/span><br \/>\n<span style=\"color: #000000\"><strong>B8.<\/strong> Dehydrate gel pieces with 500uL of 100% ACN for 15 minutes.<\/span><br \/>\n<span style=\"color: #000000\"><strong>B9.<\/strong> Remove acetonitrile and dry gel pieces in vacuum centrifuge for 10 minutes.<\/span><\/p>\n<p><span style=\"color: #000000\"><strong>Part C: Enzymatic Digestion<\/strong><\/span><\/p>\n<p style=\"padding-left: 40px\"><span style=\"color: #000000\"><strong>C1.<\/strong> Dilute enzyme stock solution with 25mM ammonium bicarbonate to obtain a 10-20<\/span><br \/>\n<span style=\"color: #000000\">ng\/uL working solution.<\/span><br \/>\n<span style=\"color: #000000\"><strong>C2.<\/strong> Add sufficient amount of the gel enzyme to cover gel pieces (typically 20-30uL), and<\/span><br \/>\n<span style=\"color: #000000\">incubate on ice for 1 hour.<\/span><br \/>\n<span style=\"color: #000000\"><strong>C3.<\/strong> Add sufficient amount of 25mM ammonium bicarbonate to cover the gel pieces.<\/span><\/p>\n<p style=\"padding-left: 80px\"><span style=\"color: #000000\"><em>The enzyme diffuses into the gel that contains the reduced and alkylated protein and acts to cleave the protein into peptides in the gel. These peptides then get extracted from the gel in the following steps. <\/em><\/span><\/p>\n<p style=\"padding-left: 40px\"><span style=\"color: #000000\"><strong>C4.<\/strong> Incubate at 37\u00b0C overnight.<\/span><\/p>\n<p><span style=\"color: #000000\"><strong>Part D: Peptide Extraction<\/strong><\/span><\/p>\n<p style=\"padding-left: 40px\"><span style=\"color: #000000\"><em>From this point forward, all supernatant collected should be transferred to a new, clean microfuge tube. Supernatant from each step can be combined with the previous step\u2019s supernatant for each individual sample.<\/em><\/span><br \/>\n<span style=\"color: #000000\"><strong>D1.<\/strong> Remove any supernatant (which will contain peptides) and transfer to a new clean tube.<\/span><\/p>\n<p style=\"padding-left: 40px\"><span style=\"color: #000000\"><strong>D2.<\/strong> Add 50uL of 5% (v\/v) formic acid, and allow gel band to incubate for 15 minutes at room temperature.<\/span><br \/>\n<span style=\"color: #000000\"><strong>D3.<\/strong> Remove formic acid and combine with previous supernatant.<\/span><br \/>\n<span style=\"color: #000000\"><strong>D4.<\/strong> Add 50uL of 100% Acetonitrile, and allow gel band to incubate for 15 minutes at room temperature.<\/span><br \/>\n<span style=\"color: #000000\"><strong>D5.<\/strong> Remove formic acid and combine with previous supernatant.<\/span><br \/>\n<span style=\"color: #000000\"><strong>D6.<\/strong> Repeat Steps D2-D5.<\/span><br \/>\n<span style=\"color: #000000\"><strong>D7.<\/strong> Dry combined supernatants using a vacuum centrifuge.<\/span><\/p>\n<p>&nbsp;<\/p>\n<p>&nbsp;<\/p>\n<p><a href=\"https:\/\/lab.research.sickkids.ca\/sparc-molecular-analysis\/wp-content\/uploads\/sites\/61\/2017\/12\/in-gel-digestion.pdf\"><span style=\"color: #307897\"><em><strong>Download and save a PDF copy of the In-Gel Digestion Protocol.\u00a0<\/strong><\/em><\/span><\/a><\/p>\n","protected":false},"excerpt":{"rendered":"<p>IN-GEL DIGESTION AND EXTRACTION PROTOCOL Solutions Required: 1% Acetic Acid 100% Acetonitrile (ACN), HPLC Grade 10mM Dithiothreitol (DTT) 55mM iodoacetamide (IAA), prepared immediately prior to use, protect from light 25mM NH4HCO3 Gel Wash Solution 25mM NH4HCO3 50% ACN Enzyme of interest Protocol: Part A: Excision &amp; De-stain of gel bands A1. Excise protein bands of&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-1811","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>In-Gel Digestion - 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\/in-gel-digestion\/\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"In-Gel Digestion\" \/>\n<meta property=\"og:description\" content=\"IN-GEL DIGESTION AND EXTRACTION PROTOCOL Solutions Required: 1% Acetic Acid 100% Acetonitrile (ACN), HPLC Grade 10mM Dithiothreitol (DTT) 55mM iodoacetamide (IAA), prepared immediately prior to use, protect from light 25mM NH4HCO3 Gel Wash Solution 25mM NH4HCO3 50% ACN Enzyme of interest Protocol: Part A: Excision &amp; De-stain of gel bands A1. Excise protein bands of&hellip;\" \/>\n<meta property=\"og:url\" content=\"https:\/\/lab.research.sickkids.ca\/sparc-molecular-analysis\/in-gel-digestion\/\" \/>\n<meta property=\"og:site_name\" content=\"SPARC BioCentre Molecular Analysis\" \/>\n<meta name=\"twitter:card\" content=\"summary_large_image\" \/>\n<meta name=\"twitter:label1\" content=\"Est. reading time\" \/>\n\t<meta name=\"twitter:data1\" content=\"3 minutes\" \/>\n<script type=\"application\/ld+json\" class=\"yoast-schema-graph\">{\"@context\":\"https:\/\/schema.org\",\"@graph\":[{\"@type\":\"WebPage\",\"@id\":\"https:\/\/lab.research.sickkids.ca\/sparc-molecular-analysis\/in-gel-digestion\/\",\"url\":\"https:\/\/lab.research.sickkids.ca\/sparc-molecular-analysis\/in-gel-digestion\/\",\"name\":\"In-Gel Digestion - SPARC BioCentre Molecular Analysis\",\"isPartOf\":{\"@id\":\"https:\/\/lab.research.sickkids.ca\/sparc-molecular-analysis\/#website\"},\"datePublished\":\"2021-09-16T15:25:08+00:00\",\"breadcrumb\":{\"@id\":\"https:\/\/lab.research.sickkids.ca\/sparc-molecular-analysis\/in-gel-digestion\/#breadcrumb\"},\"inLanguage\":\"en-US\",\"potentialAction\":[{\"@type\":\"ReadAction\",\"target\":[\"https:\/\/lab.research.sickkids.ca\/sparc-molecular-analysis\/in-gel-digestion\/\"]}]},{\"@type\":\"BreadcrumbList\",\"@id\":\"https:\/\/lab.research.sickkids.ca\/sparc-molecular-analysis\/in-gel-digestion\/#breadcrumb\",\"itemListElement\":[{\"@type\":\"ListItem\",\"position\":1,\"name\":\"Home\",\"item\":\"https:\/\/lab.research.sickkids.ca\/sparc-molecular-analysis\/\"},{\"@type\":\"ListItem\",\"position\":2,\"name\":\"In-Gel Digestion\"}]},{\"@type\":\"WebSite\",\"@id\":\"https:\/\/lab.research.sickkids.ca\/sparc-molecular-analysis\/#website\",\"url\":\"https:\/\/lab.research.sickkids.ca\/sparc-molecular-analysis\/\",\"name\":\"SPARC BioCentre Molecular Analysis\",\"description\":\"\",\"potentialAction\":[{\"@type\":\"SearchAction\",\"target\":{\"@type\":\"EntryPoint\",\"urlTemplate\":\"https:\/\/lab.research.sickkids.ca\/sparc-molecular-analysis\/?s={search_term_string}\"},\"query-input\":{\"@type\":\"PropertyValueSpecification\",\"valueRequired\":true,\"valueName\":\"search_term_string\"}}],\"inLanguage\":\"en-US\"}]}<\/script>\n<!-- \/ Yoast SEO Premium plugin. -->","yoast_head_json":{"title":"In-Gel Digestion - SPARC BioCentre Molecular Analysis","robots":{"index":"index","follow":"follow","max-snippet":"max-snippet:-1","max-image-preview":"max-image-preview:large","max-video-preview":"max-video-preview:-1"},"canonical":"https:\/\/lab.research.sickkids.ca\/sparc-molecular-analysis\/in-gel-digestion\/","og_locale":"en_US","og_type":"article","og_title":"In-Gel Digestion","og_description":"IN-GEL DIGESTION AND EXTRACTION PROTOCOL Solutions Required: 1% Acetic Acid 100% Acetonitrile (ACN), HPLC Grade 10mM Dithiothreitol (DTT) 55mM iodoacetamide (IAA), prepared immediately prior to use, protect from light 25mM NH4HCO3 Gel Wash Solution 25mM NH4HCO3 50% ACN Enzyme of interest Protocol: Part A: Excision &amp; De-stain of gel bands A1. Excise protein bands of&hellip;","og_url":"https:\/\/lab.research.sickkids.ca\/sparc-molecular-analysis\/in-gel-digestion\/","og_site_name":"SPARC BioCentre Molecular Analysis","twitter_card":"summary_large_image","twitter_misc":{"Est. reading time":"3 minutes"},"schema":{"@context":"https:\/\/schema.org","@graph":[{"@type":"WebPage","@id":"https:\/\/lab.research.sickkids.ca\/sparc-molecular-analysis\/in-gel-digestion\/","url":"https:\/\/lab.research.sickkids.ca\/sparc-molecular-analysis\/in-gel-digestion\/","name":"In-Gel Digestion - SPARC BioCentre Molecular Analysis","isPartOf":{"@id":"https:\/\/lab.research.sickkids.ca\/sparc-molecular-analysis\/#website"},"datePublished":"2021-09-16T15:25:08+00:00","breadcrumb":{"@id":"https:\/\/lab.research.sickkids.ca\/sparc-molecular-analysis\/in-gel-digestion\/#breadcrumb"},"inLanguage":"en-US","potentialAction":[{"@type":"ReadAction","target":["https:\/\/lab.research.sickkids.ca\/sparc-molecular-analysis\/in-gel-digestion\/"]}]},{"@type":"BreadcrumbList","@id":"https:\/\/lab.research.sickkids.ca\/sparc-molecular-analysis\/in-gel-digestion\/#breadcrumb","itemListElement":[{"@type":"ListItem","position":1,"name":"Home","item":"https:\/\/lab.research.sickkids.ca\/sparc-molecular-analysis\/"},{"@type":"ListItem","position":2,"name":"In-Gel Digestion"}]},{"@type":"WebSite","@id":"https:\/\/lab.research.sickkids.ca\/sparc-molecular-analysis\/#website","url":"https:\/\/lab.research.sickkids.ca\/sparc-molecular-analysis\/","name":"SPARC BioCentre Molecular Analysis","description":"","potentialAction":[{"@type":"SearchAction","target":{"@type":"EntryPoint","urlTemplate":"https:\/\/lab.research.sickkids.ca\/sparc-molecular-analysis\/?s={search_term_string}"},"query-input":{"@type":"PropertyValueSpecification","valueRequired":true,"valueName":"search_term_string"}}],"inLanguage":"en-US"}]}},"_links":{"self":[{"href":"https:\/\/lab.research.sickkids.ca\/sparc-molecular-analysis\/wp-json\/wp\/v2\/pages\/1811","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/lab.research.sickkids.ca\/sparc-molecular-analysis\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/lab.research.sickkids.ca\/sparc-molecular-analysis\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/lab.research.sickkids.ca\/sparc-molecular-analysis\/wp-json\/wp\/v2\/users\/205"}],"replies":[{"embeddable":true,"href":"https:\/\/lab.research.sickkids.ca\/sparc-molecular-analysis\/wp-json\/wp\/v2\/comments?post=1811"}],"version-history":[{"count":3,"href":"https:\/\/lab.research.sickkids.ca\/sparc-molecular-analysis\/wp-json\/wp\/v2\/pages\/1811\/revisions"}],"predecessor-version":[{"id":1814,"href":"https:\/\/lab.research.sickkids.ca\/sparc-molecular-analysis\/wp-json\/wp\/v2\/pages\/1811\/revisions\/1814"}],"wp:attachment":[{"href":"https:\/\/lab.research.sickkids.ca\/sparc-molecular-analysis\/wp-json\/wp\/v2\/media?parent=1811"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}