{"id":2292,"date":"2017-06-08T20:53:52","date_gmt":"2017-06-09T00:53:52","guid":{"rendered":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/resources\/lab-methods\/gst\/"},"modified":"2018-07-20T16:30:08","modified_gmt":"2018-07-20T20:30:08","slug":"gst","status":"publish","type":"page","link":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/resources\/lab-methods\/gst\/","title":{"rendered":"GST Fusion Protein Purification from Yeast"},"content":{"rendered":"<div>\n<p><em>A Dohlman Lab Protocol<\/em><\/p>\n<div>\n<ul>\n<li>5 ml overnight culture of your favorite yeast in your favorite medium.<\/li>\n<li>Inoculate 50 ml and grow 30o C shaking O\/N until OD600 = 0.8 to 1.2. For SCD cultures use 1\/500 and 1\/1500 dilutions.<\/li>\n<li>Optional: add alpha-factor to 2.5 \u00b5M. Continue shaking at 30o C for 60 min.<\/li>\n<li>Add 1 M NaN 3 to 10 mM (final concentration) and move cultures to ice. Everything must remain cold from here on out.<\/li>\n<li>Spin cells 3 K, 10 min at 4o C.<\/li>\n<li>Discard supe and resuspend cells with 0.5 ml 10 mM NaN3. Transfer to 1.5 ml microfuge tube (not autoclaved).<\/li>\n<li>Spin 3 K, 10 min at 4o C. Alternatively spin 8 K, 1 min at room temperature.<\/li>\n<li>Discard supe. Optional: freeze pellet at -80o C.<\/li>\n<li>Resuspend in 1 ml of cold 10 mM NaN3.<\/li>\n<li>Measure OD600. Adjust volumes so that there are an equal number of cells in each sample. A total OD600 of 30 per sample is best.<\/li>\n<li>Wash with 1 ml of Lysis Buffer.<\/li>\n<li>Spin 3 K 10 min at 4o C and discard supe.<\/li>\n<li>Resuspend in 400 ul Lysis Buffer.<\/li>\n<li>Add a scoop of glass beads to a 0.5 ml PCR tube. Transfer cell lysate to the PCR tube<\/li>\n<li>Vortex 1 min, 4X. Keep samples cold between vortexing.<\/li>\n<li>Poke a hole in the bottom of the tube and spin cell lysate in a new microfuge tube 1.5 K or 500 X g, 10 min., 4o C.<\/li>\n<li>Transfer liquid from bottom tube into a new microfuge tube.<\/li>\n<li>Spin again 1.5 K, 10 min, 4o C and again transfer liquid into a new microfuge tube.<\/li>\n<li>Add Triton X-100 to 1.5 % and rock for 60 min at 4o C.<\/li>\n<li>Spin (3 K, 10 min, 4o C) and transfer the supe to a new microfuge tube.<\/li>\n<li>Remove 30 ul of liquid and add 30 ul 2X SDS PAGE Sample buffer. This will reflect protein content before Glutathione purification.<\/li>\n<li>To the remaining liquid, add 100 ul 40 % slurry of Glutathione beads and mix at 4o C for 2 h (overnight is usually fine). Glutathione beads should be prewashed 3 X with PBS and 1 X with lysis buffer before resuspending as a 40 % slurry in lysis buffer.<\/li>\n<li>Wash glutathione beads five times with PBS, 1 % Triton X-100, 300 mM NaCl at RT. Spin 2 K, 5 min, at room temperature. Rock sample for 5 min between washes. Change tubes after the first wash to reduce nonspecific binding to the tube itself.<\/li>\n<li>Resuspend in SDS-PAGE Sample buffer.\n<ul>\n<li>Alternatively elute 3 times with 1-2 column volumes of 5-10 mM reduced glutathione, 50 mM Tris pH 8, and mix with 6X SDS-PAGE sample buffer before stripping the beads with SDS-PAGE sample buffer.<\/li>\n<\/ul>\n<\/li>\n<li>Heat to 100o C for 10 min. Then store at -20o C. Protein is ready to be run on SDS-PAGE Gel.<\/li>\n<\/ul>\n<div><strong>Lysis Buffer 20 ml<\/strong><\/div>\n<table class=\"plain\" style=\"width: 443px;\" border=\"1\" cellspacing=\"2\" cellpadding=\"0\">\n<tbody>\n<tr>\n<td width=\"34%\"><b> Stock<\/b><\/td>\n<td width=\"34%\"><b>Volume<\/b><\/td>\n<td width=\"32%\"><b>Final<\/b><\/td>\n<\/tr>\n<tr>\n<td width=\"34%\">3.3 M* Triethanolamine (pH 7.2)<\/td>\n<td width=\"34%\">194 \u00b5l *<\/td>\n<td width=\"32%\">40 mM<\/td>\n<\/tr>\n<tr>\n<td width=\"34%\">0.5 M EDTA (pH 8)<\/td>\n<td width=\"34%\">80 \u00b5l<\/td>\n<td width=\"32%\">2 mM<\/td>\n<\/tr>\n<tr>\n<td width=\"34%\">5 M NaCl<\/td>\n<td width=\"34%\">600 \u00b5l<\/td>\n<td width=\"32%\">150 mM<\/td>\n<\/tr>\n<tr>\n<td width=\"34%\">0.1 M DTT<\/td>\n<td width=\"34%\">400 \u00b5l<\/td>\n<td width=\"32%\">2 mM<\/td>\n<\/tr>\n<tr>\n<td width=\"34%\">10 mM AEBSF<\/td>\n<td width=\"34%\">0.4 ml<\/td>\n<td width=\"32%\">0.2 mM<\/td>\n<\/tr>\n<tr>\n<td width=\"34%\">1.5 mg\/ml leupeptin<\/td>\n<td width=\"34%\">200 \u00b5l<\/td>\n<td width=\"32%\">15 \u00b5g\/ml<\/td>\n<\/tr>\n<tr>\n<td width=\"34%\">0.5 mg\/ml pepstatin<\/td>\n<td width=\"34%\">20 \u00b5l<\/td>\n<td width=\"32%\">20 \u00b5g\/ml<\/td>\n<\/tr>\n<tr>\n<td width=\"34%\">1 M benzamidine<\/td>\n<td width=\"34%\">20 \u00b5l<\/td>\n<td width=\"32%\">1 mM<\/td>\n<\/tr>\n<tr>\n<td width=\"34%\">0.5 mg\/ml aprotinin<\/td>\n<td width=\"34%\">400 \u00b5l<\/td>\n<td width=\"32%\">10 \u00b5g\/ml<\/td>\n<\/tr>\n<tr>\n<td width=\"34%\">100 mM b-glycerolphosphate<\/td>\n<td width=\"34%\">20 \u00b5l<\/td>\n<td width=\"32%\">100 \u00b5M<\/td>\n<\/tr>\n<tr>\n<td width=\"34%\">50 mM Na-o-vanadate<\/td>\n<td width=\"34%\">200 \u00b5l<\/td>\n<td width=\"32%\">0.5 mM<\/td>\n<\/tr>\n<tr>\n<td width=\"34%\"><\/td>\n<td width=\"34%\">HOH to 20 mls<\/td>\n<td width=\"32%\"><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<p>NOTES<\/p>\n<p>Thanks to Paul DiBello and Jiyoung Cha for their refinements of this protocol.<\/p>\n<p>* Indicates a correction from an eariler version of the protocol.<\/p>\n<p>1. For lysis in the presense of GDP and GTP I use a final concentration of 10 uM GDP or 20 uM GTPgammaS and a final concentration of 3 mM MgCl2 in the lysis buffer.<br \/>\n2. You can substitute protease inhibitor cocktail (Sigma P8215) for individual protease inhibitors (AEBSF, leupeptin, pepstatin, benzamidine, aprotinin).<\/p>\n<p>3. For lysis to determine phosphorylation, you may wish to add more phosphatase inhibitors (in addition to beta-glycerolphosphate and Na-o-vanadate) to the lysis buffer, or you may wish to omit phosphatase inhibitors altogether:<\/p>\n<table class=\"plain\" style=\"width: 307px;\" border=\"0\" cellspacing=\"2\" cellpadding=\"0\">\n<tbody>\n<tr>\n<td width=\"47%\" height=\"23\">50mM Na-M-Vanadate<\/td>\n<td width=\"24%\">200ml<\/td>\n<td width=\"29%\">0.5mM<\/td>\n<\/tr>\n<tr>\n<td width=\"47%\" height=\"23\">100mM Na-pyrophosphate<\/td>\n<td width=\"24%\">2 ml<\/td>\n<td width=\"29%\">10mM<\/td>\n<\/tr>\n<tr>\n<td width=\"47%\" height=\"23\">2mg\/ml Phosvitin<\/td>\n<td width=\"24%\">10\u00b5l<\/td>\n<td width=\"29%\">1\u00b5g\/ml<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<\/div>\n<\/div>\n","protected":false},"excerpt":{"rendered":"<p>A Dohlman Lab Protocol 5 ml overnight culture of your favorite yeast in your favorite medium. Inoculate 50 ml and grow 30o C shaking O\/N until OD600 = 0.8 to 1.2. For SCD cultures use 1\/500 and 1\/1500 dilutions. Optional: add alpha-factor to 2.5 \u00b5M. Continue shaking at 30o C for 60 min. Add 1 &hellip; <a href=\"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/resources\/lab-methods\/gst\/\" aria-label=\"Read more about GST Fusion Protein Purification from Yeast\">Read more<\/a><\/p>\n","protected":false},"author":22429,"featured_media":0,"parent":2239,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"_acf_changed":false,"footnotes":"","_links_to":"","_links_to_target":""},"class_list":["post-2292","page","type-page","status-publish","hentry","odd"],"acf":[],"_links_to":[],"_links_to_target":[],"_links":{"self":[{"href":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/wp-json\/wp\/v2\/pages\/2292","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/wp-json\/wp\/v2\/users\/22429"}],"replies":[{"embeddable":true,"href":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/wp-json\/wp\/v2\/comments?post=2292"}],"version-history":[{"count":0,"href":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/wp-json\/wp\/v2\/pages\/2292\/revisions"}],"up":[{"embeddable":true,"href":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/wp-json\/wp\/v2\/pages\/2239"}],"wp:attachment":[{"href":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/wp-json\/wp\/v2\/media?parent=2292"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}