{"id":756,"date":"2013-09-06T17:10:49","date_gmt":"2013-09-06T16:10:49","guid":{"rendered":"http:\/\/blogs.lshtm.ac.uk\/alsfordlab\/?page_id=756"},"modified":"2014-11-26T11:30:04","modified_gmt":"2014-11-26T10:30:04","slug":"protein-preparation","status":"publish","type":"page","link":"https:\/\/blogs.lshtm.ac.uk\/alsfordlab\/protocols\/protein-preparation\/","title":{"rendered":"Protein preparation"},"content":{"rendered":"<p><em><strong>Materials<\/strong><\/em><\/p>\n<ul>\n<li>1x Sample buffer (62 mM Tris [pH 7], 10% glycerol, 2.3% SDS, 5% \u03b2-mercaptoethanol, bromophenol blue [the \u2018end of a 200 ul tip\u2019 is enough for 10 ml sample buffer]) store at -20\u00b0C<\/li>\n<\/ul>\n<p><strong><em>Protocol<\/em><\/strong><\/p>\n<ol>\n<li>Pellet an exponentially growing culture (typically, 10 ml at ~10<sup>6<\/sup> cells ml<sup>-1<\/sup>) at 1000 g for 10 minutes<\/li>\n<li>Replace the supernatant with an equal volume of PBS and repeat centrifugation (1)<\/li>\n<li>Discard the supernatant and resuspend the cell pellet in 500 \u03bcl PBS, and centrifuge in a microfuge for 2 minutes at 4500 g<\/li>\n<li>Remove as much of the supernatant as possible, and resuspend the cell pellet in <b>1x<\/b> <b>sample buffer<\/b> to a final concentration of 10<sup>5<\/sup> cells per \u03bcl<\/li>\n<li>Vortex the cell lysate for ~2 seconds and place at 100\u00b0C for 2 min; centrifuge briefly (~2 seconds), and store at -20\u00b0C for up to 3 months<\/li>\n<\/ol>\n<p><em>NB: if protein of interest is likely to be membrane-associated don&#8217;t boil, instead resuspend in <strong>1x sample buffer<\/strong> containing protease inhibitors; alternatively, follow the <a title=\"html\" href=\"http:\/\/blogs.lshtm.ac.uk\/alsfordlab\/protocols\/hypotonic-lysis\/\">hypotonic lysis<\/a> protocol to assess the distribution of your protein between soluble and membrane fractions.<\/em><\/p>\n","protected":false},"excerpt":{"rendered":"<p>Materials 1x Sample buffer (62 mM Tris [pH 7], 10% glycerol, 2.3% SDS, 5% \u03b2-mercaptoethanol,&#8230;<\/p>\n","protected":false},"author":116,"featured_media":0,"parent":17,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"footnotes":""},"class_list":["post-756","page","type-page","status-publish","hentry","odd"],"yoast_head":"<!-- This site is optimized with the Yoast SEO plugin v26.9 - 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