{"id":2301,"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\/protein-prep\/"},"modified":"2018-07-20T16:32:23","modified_gmt":"2018-07-20T20:32:23","slug":"protein-prep","status":"publish","type":"page","link":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/resources\/lab-methods\/protein-prep\/","title":{"rendered":"SDS Whole Cell Extracts"},"content":{"rendered":"<div>\n<p><em>A Dohlman Lab Protocol<\/em><\/p>\n<div>\n<p>REFERENCE: Hoffman, G., Garrison, T. R., and Dohlman, H. G., Analysis of RGS proteins in Saccharomyces cerevisiae, Methods Enzymol.344:617-631, 2002.<\/p>\n<p>-Use sterile technique and sterile solutions in steps 1 to 3.-<\/p>\n<p>1. Using a saturated starter culture, inoculate 25 to 30 ml of appropriate media in a 125 ml flask.<\/p>\n<ul>\n<li>Since it is often difficult to estimate the growth rate of yeast, it is helpful to start several 25 ml cultures, each with a different dilution of the starter culture (e.g. 1:100, 1:300, 1:900).<\/li>\n<\/ul>\n<p>2. Grow at 30 C shaking (250 rpm) until the OD600 nm ~ 1.0 (This is usually done overnight).<\/p>\n<ul>\n<li>When growing several strains at once, it is likely that they will all reach OD600 nm ~ 1.0 at different times. If desired, sodium azide (1M stock in water, diluted to a final concentration of 10 mM) can be added to a culture once it reaches an OD600 nm ~ 1.0. The culture can then be placed on ice until the others are ready.<\/li>\n<\/ul>\n<p>3. Transfer to a 50 ml conical tube and centrifuge for 10 min at 2000 x g at 4 C.<\/p>\n<p>4. Resuspend each sample in 1 ml of 10 mM sodium azide and place on ice.<\/p>\n<p>5. Calculate the volume of resuspended cells that would translate to an OD600 nm reading of 10. For example, this would equal 1 ml if 10 ml of culture at OD600 nm = 1.0 had been centrifuged and resuspended.<\/p>\n<ul>\n<li>This step is necessary to equalize the amount of cells (and protein) in a given volume of whole cell extract.<\/li>\n<\/ul>\n<p>6. Transfer the calculated volume of resuspended cells to a microfuge tube and centrifuge at 16,000 x g for 1 min.<\/p>\n<p>7. Aspirate the supernatent.<\/p>\n<p>8. Resuspend the pellet in 200 ul of 1X SDS-PAGE sample buffer.<\/p>\n<p>9. Immediately place in a 100 C heat block for 10 min.<\/p>\n<p>10. Allow the tube to cool and add 200 ul of glass beads (Sigma, #G-8772).<\/p>\n<p>11. Vortex at high speed for 2 min. Invert after the first min.<\/p>\n<ul>\n<li>Several tubes can be vortexed at the same time by using a foam tube floater to hold them together.<\/li>\n<\/ul>\n<p>12. Using a 21 gauge needle, poke a hole in the bottom of each tube and place it into a new microfuge tube.<\/p>\n<p>13. Centrifuge at 2000 x g for 10 sec to expel the liquid into the bottom tube, leaving the glass beads in the top tube.<\/p>\n<p>14. Discard the glass beads and centrifuge the bottom tube at 16,000 x g for 2 min. This sediments any insoluble material.<\/p>\n<p>15. Transfer the supernatant to a new microfuge tube. Store at -20 C.<\/p>\n<p>16. When ready to use, heat at 37 C for 10 min, vortex, and centrifuge at 16,000 x g for 1 min.<\/p>\n<ul>\n<li>Keep in mind that repeated freezing and thawing can degrade the protein sample.<\/li>\n<\/ul>\n<p>17. Immunoblots can be performed using standard methods.<\/p>\n<p>Updated 01\/23\/02<\/p>\n<\/div>\n<\/div>\n","protected":false},"excerpt":{"rendered":"<p>A Dohlman Lab Protocol REFERENCE: Hoffman, G., Garrison, T. R., and Dohlman, H. G., Analysis of RGS proteins in Saccharomyces cerevisiae, Methods Enzymol.344:617-631, 2002. -Use sterile technique and sterile solutions in steps 1 to 3.- 1. Using a saturated starter culture, inoculate 25 to 30 ml of appropriate media in a 125 ml flask. Since &hellip; <a href=\"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/resources\/lab-methods\/protein-prep\/\" aria-label=\"Read more about SDS Whole Cell Extracts\">Read more<\/a><\/p>\n","protected":false},"author":22429,"featured_media":0,"parent":2239,"menu_order":5,"comment_status":"closed","ping_status":"closed","template":"","meta":{"_acf_changed":false,"footnotes":"","_links_to":"","_links_to_target":""},"class_list":["post-2301","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\/2301","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=2301"}],"version-history":[{"count":0,"href":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/wp-json\/wp\/v2\/pages\/2301\/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=2301"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}