{"id":2293,"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\/haloassay\/"},"modified":"2018-07-20T16:31:49","modified_gmt":"2018-07-20T20:31:49","slug":"haloassay","status":"publish","type":"page","link":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/resources\/lab-methods\/haloassay\/","title":{"rendered":"Pheromone Halo Assay"},"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 throughout this method.-<\/p>\n<p>1. Grow a starter culture at 30 C with shaking (250 rpm) until it reaches saturation.<\/p>\n<ul>\n<li>It is important to make sure all strains to be tested are completely saturated, since cell density can affect the size of the halo.<\/li>\n<\/ul>\n<p>2. Microwave a sterilized solution of 0.5 % agar until melted and place in a 55 C water bath.<\/p>\n<ul>\n<li>Don\u2019t leave unattended when microwaving since the agar tends to boil over suddenly.<\/li>\n<li>Make sure the agar equilibrates to 55 C to 60 C before starting the assay; otherwise it will kill the cells.<\/li>\n<\/ul>\n<p>3. Distribute several paper disks (Difco, #1599-33-6) into a sterile petri dish, and spot either 5 or 15 of synthetic alpha-factor (1 \u2013 5 mg\/ml) onto each disk.<\/p>\n<ul>\n<li>In some instances, it may be beneficial to use more disks per plate to obtain a wider range of alpha-factor concentrations.<\/li>\n<li>This should be done no more than 1 to 2 hrs before use.<\/li>\n<\/ul>\n<p>4. Aliquot 4 ml of 0.5 % agar into a 5 or 14 ml plastic tube with a pop-off cap (e.g. Falcon, #35-2063).<\/p>\n<p>5. Immediately before performing the assay, transfer a small volume of the saturated starter culture into the tube containing agar. 100 ul of starter culture should be used for strains grown in SCD; 10 ul should be used for strains grown in YPD.<\/p>\n<p>6. Invert a few times, and pour onto a pre-warmed plate containing the appropriate solid media. Swirl to cover the plate evenly. This step should be done quickly to prevent the agar from solidifying.<\/p>\n<ul>\n<li>It is convenient to do two tubes at a time, one in each hand.<\/li>\n<li>Bubbles can usually be eliminated by poking with flamed sterile forceps.<\/li>\n<\/ul>\n<p>7. On the plate, place one paper disk containing 5 ul alpha-factor, and one disk containing 15 ul alpha-factor.<\/p>\n<ul>\n<li>When doing several assays, it is helpful to use a paper template for consistent placement of the disks on each plate.<\/li>\n<\/ul>\n<p>8. Place the plate at 30 C until a lawn of cells appears. The halo assay will result in a zone of growth inhibition surrounding the filters soaked with pheromone, as seen in the example below. Halos can usually be seen clearly after 24 hrs for most strains.<\/p>\n<ul>\n<li>It is often desirable to let the cells grow longer (up to several days) to get a denser lawn and to observe any changes that might occur over an extended period of time.<\/li>\n<\/ul>\n<p>9. Differences in halo size are normally big enough to be detected by eye.<\/p>\n<ul>\n<li>It is also possible to measure halo size and plot this value vs. log [alpha-factor].<\/li>\n<\/ul>\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 throughout this method.- 1. Grow a starter culture at 30 C with shaking (250 rpm) until it reaches saturation. It is important to make sure &hellip; <a href=\"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/resources\/lab-methods\/haloassay\/\" aria-label=\"Read more about Pheromone Halo Assay\">Read more<\/a><\/p>\n","protected":false},"author":22429,"featured_media":0,"parent":2239,"menu_order":4,"comment_status":"closed","ping_status":"closed","template":"","meta":{"_acf_changed":false,"footnotes":"","_links_to":"","_links_to_target":""},"class_list":["post-2293","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\/2293","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=2293"}],"version-history":[{"count":0,"href":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/wp-json\/wp\/v2\/pages\/2293\/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=2293"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}