High-throughput ß-galactosidase and ß-glucuronidase Assays Using Fluorogenic Substrates
β-galactosidase and β-glucuronidase enzymes are commonly used as reporters for gene expression from gene promoter-lacZ or uidA fusions (respectively). The protocol described here is a high-throughput alternative to the commonly used Miller assay (Miller, 1972) that utilises a fluorogenic substrate (...
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| Format: | Journal Article |
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2013
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| Online Access: | http://hdl.handle.net/20.500.11937/46320 |
| _version_ | 1848757524637941760 |
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| author | Ramsay, Joshua |
| author_facet | Ramsay, Joshua |
| author_sort | Ramsay, Joshua |
| building | Curtin Institutional Repository |
| collection | Online Access |
| description | β-galactosidase and β-glucuronidase enzymes are commonly used as reporters for gene expression from gene promoter-lacZ or uidA fusions (respectively). The protocol described here is a high-throughput alternative to the commonly used Miller assay (Miller, 1972) that utilises a fluorogenic substrate (Fiksdal et al., 1994) and 96-well plate format. The fluorogenic substrates 4-Methylumbelliferyl β-D-galactoside (for β-galactosidase assays) (Ramsay et al., 2013) or 4-Methylumbelliferyl β-D-glucuronide (for β-glucuronidase assays) (Ramsay et al., 2011) are cleaved to produce the fluorescent product 4-methylumbelliferone. Cells are permeabilized by freeze-thawing and lysozyme, and the production of 4-methylumbelliferone is monitored continuously by a fluorescence microplate reader as a kinetic assay. The rate of increase in fluorescence is then calculated, from which relative gene-expression levels are extrapolated. Due to the high sensitivity fluorescence-based detection of 4-methylumbelliferone and the high density of time points collected, this assay may offer increased accuracy in the quantification of low-level gene expression. The assay requires small sample volumes and minimal preparation time. The permeabilisation conditions outlined in this protocol have been optimised for Gram-negative bacteria (specifically Escherichia coli and Serratia), but is likely suitable for other organisms with minimal optimisation. |
| first_indexed | 2025-11-14T09:29:28Z |
| format | Journal Article |
| id | curtin-20.500.11937-46320 |
| institution | Curtin University Malaysia |
| institution_category | Local University |
| last_indexed | 2025-11-14T09:29:28Z |
| publishDate | 2013 |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | curtin-20.500.11937-463202017-09-13T15:34:51Z High-throughput ß-galactosidase and ß-glucuronidase Assays Using Fluorogenic Substrates Ramsay, Joshua β-galactosidase and β-glucuronidase enzymes are commonly used as reporters for gene expression from gene promoter-lacZ or uidA fusions (respectively). The protocol described here is a high-throughput alternative to the commonly used Miller assay (Miller, 1972) that utilises a fluorogenic substrate (Fiksdal et al., 1994) and 96-well plate format. The fluorogenic substrates 4-Methylumbelliferyl β-D-galactoside (for β-galactosidase assays) (Ramsay et al., 2013) or 4-Methylumbelliferyl β-D-glucuronide (for β-glucuronidase assays) (Ramsay et al., 2011) are cleaved to produce the fluorescent product 4-methylumbelliferone. Cells are permeabilized by freeze-thawing and lysozyme, and the production of 4-methylumbelliferone is monitored continuously by a fluorescence microplate reader as a kinetic assay. The rate of increase in fluorescence is then calculated, from which relative gene-expression levels are extrapolated. Due to the high sensitivity fluorescence-based detection of 4-methylumbelliferone and the high density of time points collected, this assay may offer increased accuracy in the quantification of low-level gene expression. The assay requires small sample volumes and minimal preparation time. The permeabilisation conditions outlined in this protocol have been optimised for Gram-negative bacteria (specifically Escherichia coli and Serratia), but is likely suitable for other organisms with minimal optimisation. 2013 Journal Article http://hdl.handle.net/20.500.11937/46320 10.21769/BioProtoc.827 fulltext |
| spellingShingle | Ramsay, Joshua High-throughput ß-galactosidase and ß-glucuronidase Assays Using Fluorogenic Substrates |
| title | High-throughput ß-galactosidase and ß-glucuronidase Assays Using Fluorogenic Substrates |
| title_full | High-throughput ß-galactosidase and ß-glucuronidase Assays Using Fluorogenic Substrates |
| title_fullStr | High-throughput ß-galactosidase and ß-glucuronidase Assays Using Fluorogenic Substrates |
| title_full_unstemmed | High-throughput ß-galactosidase and ß-glucuronidase Assays Using Fluorogenic Substrates |
| title_short | High-throughput ß-galactosidase and ß-glucuronidase Assays Using Fluorogenic Substrates |
| title_sort | high-throughput ß-galactosidase and ß-glucuronidase assays using fluorogenic substrates |
| url | http://hdl.handle.net/20.500.11937/46320 |