{"id":250,"date":"2012-04-10T16:34:39","date_gmt":"2012-04-10T16:34:39","guid":{"rendered":"https:\/\/wordpress.clarku.edu\/debrobertson\/?page_id=250"},"modified":"2012-05-23T17:19:01","modified_gmt":"2012-05-23T17:19:01","slug":"purification-of-his-tagged-luciferase","status":"publish","type":"page","link":"https:\/\/wordpress.clarku.edu\/debrobertson\/laboratory-protocols\/purification-of-his-tagged-luciferase\/","title":{"rendered":"PURIFICATION OF HIS-TAGGED LUCIFERASE"},"content":{"rendered":"

<\/center>Date: _____________<\/p>\n

Cells collected on _______________<\/p>\n

    \n
  1. Resuspend pellet in 1 mL of EXTRACTION BUFFER (EB) for every 50 mL of culture harvested.<\/li>\n
  2. Add lysozyme to EB, prior to resuspending pellets, to a final concentration of 1 mg\/mL<\/li>\n
  3. Incubate on ice for 30 min.<\/li>\n
  4. Vortex cells. Followed by 6 x 10 sec bursts of sonication with 10 sec cooling periods in between bursts.<\/li>\n
  5. Add RNAse A (10 \u00b5g\/mL) and DNase I (5 \u00b5g\/mL) and incubate on ice for 10-15 min.<\/li>\n
  6. Centrifuge lysate at 10,000 x g for 20-30 min at 4 C<\/li>\n
  7. Add ______ mL of 50% Ni-NTA slurry (equilibrated in EB). Mix gently at 4 C for 60 min.<\/li>\n
  8. Load column and wash with 10 bed volumes of WASH BUFFER #1<\/li>\n
  9. Wash with 5 bed volumes of WASH BUFFER #2<\/li>\n
  10. Elute with 5 x 1 bed volume of ELUTION BUFFER.<\/li>\n
  11. Remove 200 uL aliquot from each fraction for protein assays and store remaining fractions at -80 C.<\/li>\n<\/ol>\n

     <\/p>\n

    <\/p>\n

    BUFFERS<\/h2>\n

    <\/center><\/p>\n

    \n

    EXTRACTION BUFFER:<\/p>\n

    50 mM NaH2PO4 pH 8.0<\/p>\n

    300 mM NaCl<\/p>\n

    20 mM Imidazole<\/p>\n

    20% (v\/v) glycerol<\/p>\n

    Pefabloc<\/p><\/blockquote>\n

    WASH BUFFER #1<\/p>\n

    50 mM NaH2PO4 pH 8.0<\/p>\n

    300 mM NaCl<\/p>\n

    40 mM Imidazole<\/p>\n

    20% glycerol<\/p><\/blockquote>\n

    WASH BUFFER #2<\/p>\n

    50 mM NaH2PO4 pH 8.0<\/p>\n

    300 mM NaCl<\/p>\n

    60 mM Imidazole<\/p>\n

    20% glycerol<\/p><\/blockquote>\n

    ELUTION BUFFER<\/p>\n

    50 mM NaH2PO4 pH 8.0<\/p>\n

    300 mM NaCl<\/p>\n

    250 mM Imidazole<\/p>\n

    20% glycerol<\/p><\/blockquote>\n","protected":false},"excerpt":{"rendered":"

    Date: _____________ Cells collected on _______________ Resuspend pellet in 1 mL of EXTRACTION BUFFER (EB) for every 50 mL of culture harvested. Add lysozyme to EB, prior to resuspending pellets, to a final concentration of 1 mg\/mL Incubate on ice …<\/p>\n

    PURIFICATION OF HIS-TAGGED LUCIFERASE<\/span> Read More »<\/a><\/p>\n","protected":false},"author":72,"featured_media":0,"parent":8,"menu_order":60,"comment_status":"closed","ping_status":"closed","template":"onecolumn-page.php","meta":{"ngg_post_thumbnail":0,"footnotes":""},"class_list":["post-250","page","type-page","status-publish","hentry"],"_links":{"self":[{"href":"https:\/\/wordpress.clarku.edu\/debrobertson\/wp-json\/wp\/v2\/pages\/250","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/wordpress.clarku.edu\/debrobertson\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/wordpress.clarku.edu\/debrobertson\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/wordpress.clarku.edu\/debrobertson\/wp-json\/wp\/v2\/users\/72"}],"replies":[{"embeddable":true,"href":"https:\/\/wordpress.clarku.edu\/debrobertson\/wp-json\/wp\/v2\/comments?post=250"}],"version-history":[{"count":0,"href":"https:\/\/wordpress.clarku.edu\/debrobertson\/wp-json\/wp\/v2\/pages\/250\/revisions"}],"up":[{"embeddable":true,"href":"https:\/\/wordpress.clarku.edu\/debrobertson\/wp-json\/wp\/v2\/pages\/8"}],"wp:attachment":[{"href":"https:\/\/wordpress.clarku.edu\/debrobertson\/wp-json\/wp\/v2\/media?parent=250"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}