{"id":734,"date":"2013-09-06T13:13:52","date_gmt":"2013-09-06T12:13:52","guid":{"rendered":"http:\/\/blogs.lshtm.ac.uk\/alsfordlab\/?page_id=734"},"modified":"2014-11-26T11:30:03","modified_gmt":"2014-11-26T10:30:03","slug":"hypotonic-lysis","status":"publish","type":"page","link":"https:\/\/blogs.lshtm.ac.uk\/alsfordlab\/protocols\/hypotonic-lysis\/","title":{"rendered":"Hypotonic lysis"},"content":{"rendered":"<p>Adapted from Leung <em>et al<\/em> (2008) <a title=\"PubMed\" href=\"http:\/\/www.ncbi.nlm.nih.gov\/pubmed\/18637903\" target=\"_blank\"><i>Traffic<\/i> <b>9<\/b>:1698<\/a><\/p>\n<p>For the separation of soluble and membrane fractions, enabling the initial characterisation of membrane-associated proteins.<\/p>\n<p><em><b>Materials<\/b><\/em><\/p>\n<ul>\n<li>Phosphate Buffered Saline (PBS)<\/li>\n<li>Hypotonic Lysis Buffer (10 mM Tris-HCl, pH 7.5)<\/li>\n<li>Sample Lysis Buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40)<\/li>\n<li>2x Sample Buffer (124 mM Tris-Cl, pH6.8, 4.6% SDS, 10% \u03b2-mercaptoethanol, 20% glycerol, bromophenol blue)<\/li>\n<\/ul>\n<p><em>NB: Lysis and sample buffers should contain protease inhibitors \u2013 this precludes the need to boil the samples (boiling can interfere with SDS-PAGE fractionation of membrane-associated proteins and should, therefore, be avoided)<\/em><\/p>\n<p><em><b>Protocol<\/b><\/em><\/p>\n<ol>\n<li>Wash 1&#215;10<sup>8<\/sup> cells two times in PBS (1000g 10 minutes [~25ml], then 4500g 1 minute [~1ml] in a microfuge)<\/li>\n<li>Resuspend in 100 \u00b5l of COLD hypotonic lysis buffer and incubate on ice 5 minutes<\/li>\n<li>Centrifuge for 10 minutes in a microfuge at top speed, 4C (cold room)<\/li>\n<li>Transfer supernatant to a new eppendorf and add an equal volume of 2x sample buffer (<b>soluble fraction<\/b>)<\/li>\n<li>Resuspend pellet with 100 \u00b5l cold hypotonic lysis buffer and centrifuge as above<\/li>\n<li>Transfer supernatant to a new eppendorf and add an equal volume of 2x sample buffer (<b>wash<\/b>)<\/li>\n<li>Resuspend pellet in 100 \u00b5l of ice cold 1x sample lysis buffer and incubate on ice 25 minutes to lyse membranes. Add 2x sample buffer (<b>membrane fraction<\/b>)<\/li>\n<\/ol>\n<p>Store all samples at -20\u00b0C.<\/p>\n<p>Fractionate samples by SDS-PAGE (prior to loading vortex for ~5 sec to reduce the viscosity due to the presence of DNA).<\/p>\n","protected":false},"excerpt":{"rendered":"<p>Adapted from Leung et al (2008) Traffic 9:1698 For the separation of soluble and membrane&#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-734","page","type-page","status-publish","hentry","odd"],"yoast_head":"<!-- This site is optimized with the Yoast SEO plugin v26.9 - https:\/\/yoast.com\/product\/yoast-seo-wordpress\/ -->\n<title>Hypotonic lysis - Alsford Lab<\/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:\/\/blogs.lshtm.ac.uk\/alsfordlab\/protocols\/hypotonic-lysis\/\" \/>\n<meta property=\"og:locale\" content=\"en_GB\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"Hypotonic lysis - 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