{"id":2076,"date":"2021-07-30T11:09:53","date_gmt":"2021-07-30T11:09:53","guid":{"rendered":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/?page_id=2076"},"modified":"2021-07-30T11:25:08","modified_gmt":"2021-07-30T11:25:08","slug":"15n-13c-labeling-of-proteins-in-e-coli","status":"publish","type":"page","link":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/protocols\/15n-13c-labeling-of-proteins-in-e-coli\/","title":{"rendered":"15N\/13C labeling of proteins in E. coli"},"content":{"rendered":"\n
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Before starting:<\/h3>\n\n\n\n
  • Prepare all required reagents and solutions<\/li><\/ul>\n\n\n\n

    Protocol:<\/h3>\n\n\n\n
    1. Transform the vector into an appropriate E. coli<\/em> expression strain and plate out on minimal medium plates; incubate overnight at 37\u00b0C<\/li>
    2. Pick a colony and use this to inoculate 5 ml of Medium A; grow overnight at 37\u00b0C<\/li>
    3. Add the overnight culture to 1 liter of Medium A and grow the culture at the appropriate temperature until the OD600<\/sub> ~ 0.8 \u2013 1.0<\/li>
    4. Induce expression of the target protein and continue culturing for 2-12 hours<\/li>
    5. Harvest the cells by centrifugation at 4\u00b0C<\/li>
    6. When you don\u2019t continue immediately with the protein purification, freeze the cell pellet and store at -20\u00b0C until further usage<\/li><\/ol>\n\n\n\n

      Comment: the growth rate of the culture and the optimal induction conditions can vary significantly depending on the vector backbone, the construct design and the solubility of the protein of interest<\/p>\n\n\n\n

      Medium A (per liter):<\/h4>\n\n\n\n
      • 100 ml M9 medium (10x)<\/li>
      • 10 ml trace elements solution (100x)<\/li>
      • 20 ml 10% (w\/v) 13<\/strong><\/sup>C-glucose<\/strong><\/li>
      • 1 ml 1M MgSO4<\/sub><\/li>
      • 0.3 ml 1M CaCl2<\/sub><\/li>
      • 1 ml biotin (1 mg\/ml)<\/li>
      • 1 ml thiamin (1 mg\/ml)<\/li>
      • Appropriate antibiotics<\/li><\/ul>\n\n\n\n

        10x M9 medium (per liter)<\/h4>\n\n\n\n
        • 60 g Na2<\/sub>HPO4<\/sub><\/li>
        • 30 g KH2<\/sub>PO4<\/sub><\/li>
        • 5 g NaCl<\/li>
        • 5 g N<\/strong>15<\/strong><\/sup>H<\/strong>4<\/strong><\/sub>Cl<\/strong><\/li><\/ul>\n\n\n\n

          100 x trace elements solution (per liter):<\/h4>\n\n\n\n
          • 5 g EDTA<\/li>
          • 0.83 g FeCl3<\/sub>.6H2<\/sub>O<\/li>
          • 84 mg ZnCl2<\/sub><\/li>
          • 13 mg CuCl2<\/sub>.2H2<\/sub>O<\/li>
          • 10 mg CoCl2<\/sub>.6H2<\/sub>O<\/li>
          • 10 mg H3<\/sub>BO3<\/sub><\/li>
          • 1.6 mg MnCl2<\/sub>.6H2<\/sub>O<\/li><\/ul>\n\n\n\n

            Comment: First dissolve the 5 g EDTA in 800 ml water and adjust the pH to 7.5. Then add the other components and adjust the volume to 1 L. Sterilize the solution by filtration through a 0.22 \u00b5m filter.<\/p>\n\n\n\n

            Stock solutions<\/h4>\n\n\n\n
            • 10% (w\/v) 13<\/strong><\/sup>C-glucose<\/strong> (sterilized)<\/li>
            • 1M MgSO4 <\/sub>(sterilized)<\/li>
            • 1M CaCl2<\/sub> (sterilized)<\/li>
            • 1 mg\/ml Biotin (filter sterilized)<\/li>
            • 1 mg\/ml Thiamin (filter sterilized)<\/li><\/ul>\n\n<\/div>\n<\/div>\n\n\n
              \n
              \n\n<\/div>\n<\/div>\n<\/div>\n","protected":false},"excerpt":{"rendered":"","protected":false},"author":1,"featured_media":0,"parent":2060,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"template-title-left-aligned.php","meta":{"_acf_changed":false,"footnotes":""},"embl_taxonomy":[],"class_list":["post-2076","page","type-page","status-publish","hentry"],"acf":[],"embl_taxonomy_terms":[],"_links":{"self":[{"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/pages\/2076","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/comments?post=2076"}],"version-history":[{"count":3,"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/pages\/2076\/revisions"}],"predecessor-version":[{"id":2126,"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/pages\/2076\/revisions\/2126"}],"up":[{"embeddable":true,"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/pages\/2060"}],"wp:attachment":[{"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/media?parent=2076"}],"wp:term":[{"taxonomy":"embl_taxonomy","embeddable":true,"href":"https:\/\/www.embl.org\/groups\/protein-expression-purification\/wp-json\/wp\/v2\/embl_taxonomy?post=2076"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}