{"id":1816,"date":"2021-09-16T15:30:13","date_gmt":"2021-09-16T15:30:13","guid":{"rendered":"https:\/\/lab.research.sickkids.ca\/sparc-molecular-analysis\/?page_id=1816"},"modified":"2021-09-16T15:30:13","modified_gmt":"2021-09-16T15:30:13","slug":"in-solution-digestion","status":"publish","type":"page","link":"https:\/\/lab.research.sickkids.ca\/sparc-molecular-analysis\/in-solution-digestion\/","title":{"rendered":"In-Solution Digestion"},"content":{"rendered":"

PROTOCOL <\/strong><\/span>
\nIN-SOLUTION DIGESTION OF PROTEINS<\/strong><\/span><\/p>\n

This protocol can be used to digest proteins prior to Mass Spectrometry Analysis. <\/em><\/span><\/p>\n

Procedure:<\/strong><\/span><\/p>\n

    \n
  1. In general, proteins require denaturation and disulfide bond cleavage before enzymatic digestion can go to completion.<\/span><\/li>\n
  2. Dissolve 1\u201310mg of the target protein in 8M urea, 50mM Tris-HCl (pH 8), 4mM DTT in a reaction volume of up to 1ml (25\u00b5l minimum). If your sample does not require denaturation (i.e. cell lysate, immunoprecipitations), then re-suspend your sample in 50-100\u00b5L of 50mM NH4HCO3 (pH=8.3).<\/span><\/li>\n
  3. Add DTT to final volume of 10 mM.<\/span><\/li>\n
  4. Heat at 60\u00b0C for 30 minutes.<\/span><\/li>\n
  5. Allow sample to return to room temperature.<\/span><\/li>\n
  6. Add iodoacetamide (made fresh in ddH2O) to a final volume of 10mM.<\/span><\/li>\n
  7. Incubate at room temperature in the dark for 15 minutes.<\/span><\/li>\n
  8. Inactivate any remaining iodoacetamide by adding DTT to a final concentration of 40mM.<\/span><\/li>\n
  9. If 8M urea buffer was used, dilute out with 50mM NH4HCO3 until concentration is <1M urea.<\/span><\/li>\n
  10. Check the pH of your solution \u2013 it should be around 7.5-8.5.<\/span><\/li>\n
  11. Add modified trypsin (MS grade) to a final protease:protein ratio of 1:50-1:100 (w\/w).<\/span><\/li>\n
  12. Digest samples O\/N @ 37\u00b0C.<\/span><\/li>\n
  13. Lyophilize samples, or freeze and drop off at the SPARC facility.<\/span><\/li>\n<\/ol>\n

     <\/p>\n

     <\/p>\n

    Download and save a PDF copy of the in-solution protocol.\u00a0<\/strong><\/em><\/span><\/a><\/p>\n","protected":false},"excerpt":{"rendered":"

    PROTOCOL IN-SOLUTION DIGESTION OF PROTEINS This protocol can be used to digest proteins prior to Mass Spectrometry Analysis. Procedure: In general, proteins require denaturation and disulfide bond cleavage before enzymatic digestion can go to completion. Dissolve 1\u201310mg of the target protein in 8M urea, 50mM Tris-HCl (pH 8), 4mM DTT in a reaction volume of…<\/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-1816","page","type-page","status-publish","hentry","description-off"],"yoast_head":"\nIn-Solution 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-solution-digestion\/\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"In-Solution Digestion\" \/>\n<meta property=\"og:description\" content=\"PROTOCOL IN-SOLUTION DIGESTION OF PROTEINS This protocol can be used to digest proteins prior to Mass Spectrometry Analysis. 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Dissolve 1\u201310mg of the target protein in 8M urea, 50mM Tris-HCl (pH 8), 4mM DTT in a reaction volume of…\" \/>\n<meta property=\"og:url\" content=\"https:\/\/lab.research.sickkids.ca\/sparc-molecular-analysis\/in-solution-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=\"1 minute\" \/>\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-solution-digestion\/\",\"url\":\"https:\/\/lab.research.sickkids.ca\/sparc-molecular-analysis\/in-solution-digestion\/\",\"name\":\"In-Solution Digestion - SPARC BioCentre Molecular Analysis\",\"isPartOf\":{\"@id\":\"https:\/\/lab.research.sickkids.ca\/sparc-molecular-analysis\/#website\"},\"datePublished\":\"2021-09-16T15:30:13+00:00\",\"breadcrumb\":{\"@id\":\"https:\/\/lab.research.sickkids.ca\/sparc-molecular-analysis\/in-solution-digestion\/#breadcrumb\"},\"inLanguage\":\"en-US\",\"potentialAction\":[{\"@type\":\"ReadAction\",\"target\":[\"https:\/\/lab.research.sickkids.ca\/sparc-molecular-analysis\/in-solution-digestion\/\"]}]},{\"@type\":\"BreadcrumbList\",\"@id\":\"https:\/\/lab.research.sickkids.ca\/sparc-molecular-analysis\/in-solution-digestion\/#breadcrumb\",\"itemListElement\":[{\"@type\":\"ListItem\",\"position\":1,\"name\":\"Home\",\"item\":\"https:\/\/lab.research.sickkids.ca\/sparc-molecular-analysis\/\"},{\"@type\":\"ListItem\",\"position\":2,\"name\":\"In-Solution 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-Solution 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-solution-digestion\/","og_locale":"en_US","og_type":"article","og_title":"In-Solution Digestion","og_description":"PROTOCOL IN-SOLUTION DIGESTION OF PROTEINS This protocol can be used to digest proteins prior to Mass Spectrometry Analysis. 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