{"id":2288,"date":"2017-06-08T20:53:51","date_gmt":"2017-06-09T00:53:51","guid":{"rendered":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/resources\/lab-methods\/bgal\/"},"modified":"2018-07-20T16:31:13","modified_gmt":"2018-07-20T20:31:13","slug":"bgal","status":"publish","type":"page","link":"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/resources\/lab-methods\/bgal\/","title":{"rendered":"Liquid Beta-galactosidase Reporter Gene 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>1. Grow a starter culture at 30 C shaking (250 rpm) until it reaches saturation.<\/p>\n<ul>\n<li>The cells should be transformed with a yeast expression vector containing the lacZ gene under the control of the FUS1 promoter. Alternately, a strain can be used with the FUS1-lacZ reporter integrated into the genome. These materials are available from a number of yeast labs, including our own.<\/li>\n<\/ul>\n<p>2. Using the saturated starter culture, inoculate 5 to 25 ml of the appropriate media.<\/p>\n<ul>\n<li>Rarely, a transformed colony will not respond at all to pheromone. The cause is not known. To eliminate these \u201cflatliner\u201d strains pick several different colonies. Two hours after re-inoculation take an aliquot of each and perform a small scale assay (plus and minus 1 dose of alpha-factor). Discard any that fail to change color after substrate addition.<\/li>\n<\/ul>\n<p>3. Grow at 30 C shaking (250 rpm) until the OD600 nm ~ 0.8 (this is usually done overnight).<\/p>\n<ul>\n<li>This is the trickiest part of the assay, since it is difficult to get different strains to reach OD600 nm ~ 0.8 at the same time. The best way to handle this is to start a 2 ml starter culture about 3-4 days before the assay. The night before the assay, start a 10 to 25 ml intermediate culture. The morning of the assay, measure the absorbance of all of the intermediate cultures and dilute them down to an OD600 nm of 0.2 in pre-warmed media. The strains will now only have to go through two doublings and the amount of variance between them should be reduced. If the strains still reach OD600 nm ~ 0.8 at different times, it is acceptable to put strains which have reached OD600 nm ~ 0.8 on ice while waiting for the others.<\/li>\n<li>Re-check the absorbance of all cultures before proceeding.<\/li>\n<\/ul>\n<p>4. Aliquot 10 ul of alpha-factor at the appropriate concentrations into 96 well plates.<\/p>\n<ul>\n<li>Each strain should be tested with 8 to 10 different concentrations of alpha-factor in triplicate.<\/li>\n<li>A good set of final alpha-factor concentrations for testing an sst2delta mutant strain is: 0 , 0.00003 uM, 0.0001 uM\u2026 0.3 uM. For strains expressing a mammalian or yeast RGS protein, the final concentrations should be 100-300 fold higher. The concentration of alpha-factor added to each well must be 10-fold higher than the desired final concentration.<\/li>\n<li>Keep a frozen stock of alpha-factor and make a new set of dilutions each time the assay is performed, since alpha-factor is subject to degradation upon repeated freeze-thaw cycles or exposure to room temperature.<\/li>\n<li>A multi-channel pipettor and a plastic pipettor basin (Fisher Scientific, #13681100) is helpful for aliquoting solutions.<\/li>\n<\/ul>\n<p>5. Add 90 ul of cells to each well.<\/p>\n<p>6. Incubate 90 min at 30 C, shaking gently.<\/p>\n<p>7. During the incubation, prepare the FDG solution.<\/p>\n<p style=\"padding-left: 30px;\">Solution #1: 1 mM FDG stock diluted in 25 mM PIPES (pH 7.2)<\/p>\n<p style=\"padding-left: 30px;\">Solution #2: 0.5% Triton X-100 diluted in 250 mM PIPES (pH 7.2)<\/p>\n<ul>\n<li>Mix Solution #1 and Solution #2 in equal amounts just prior to use, and pour into a clean pipettor basin.<\/li>\n<\/ul>\n<p style=\"padding-left: 30px;\">FDG Stock: 10 mM FDG (Fluorescein di-b-D-galactopyranoside, Molecular Probes, #F-1179) in dimethyl sulfoxide (DMSO) (FDG should be stored in DMSO at -20 C; it is more stable in solution.)<\/p>\n<p>8. After the 90 min incubation, add 20 ul FDG solution per well. Shake plates gently and briefly.<\/p>\n<p>9. Cover in aluminum foil and incubate at 37 C until a bright yellow color appears in some of the wells.<\/p>\n<ul>\n<li>This can take from 10 to 90 min. Do not incubate longer than 90 min.<\/li>\n<\/ul>\n<p>10. Stop the reaction by adding 20 ul of 1 M Na2CO3 per well. Shake the plates gently and briefly.<\/p>\n<p>11. Read the plates with a fluorescence multi-well plate reader using an excitation of 485 nm and an emission of 530 nm.<\/p>\n<p>12. Read the absorbance and normalize for cell density.<\/p>\n<ul>\n<li>Alternately, use the final absorbance values [obtained immediately before aliquoting cells into the 96 well plate (step 3)] to normalize for cell density.<\/li>\n<\/ul>\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. 1. Grow a starter culture at 30 C shaking (250 rpm) until it reaches saturation. The cells should be transformed with a yeast expression vector containing the lacZ gene under &hellip; <a href=\"https:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/resources\/lab-methods\/bgal\/\" aria-label=\"Read more about Liquid Beta-galactosidase Reporter Gene Assay\">Read more<\/a><\/p>\n","protected":false},"author":22429,"featured_media":0,"parent":2239,"menu_order":3,"comment_status":"closed","ping_status":"closed","template":"","meta":{"_acf_changed":false,"footnotes":"","_links_to":"","_links_to_target":""},"class_list":["post-2288","page","type-page","status-publish","hentry","odd"],"acf":[],"yoast_head":"<!-- This site is optimized with the Yoast SEO plugin v26.8 - https:\/\/yoast.com\/product\/yoast-seo-wordpress\/ -->\n<title>Liquid Beta-galactosidase Reporter Gene Assay - Dohlman 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:\/\/www.med.unc.edu\/pharm\/dohlmanlab\/resources\/lab-methods\/bgal\/\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"Liquid Beta-galactosidase Reporter Gene Assay - Dohlman Lab\" \/>\n<meta property=\"og:description\" content=\"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. 1. Grow a starter culture at 30 C shaking (250 rpm) until it reaches saturation. 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