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    High-throughput ß-galactosidase and ß-glucuronidase Assays Using Fluorogenic Substrates

    240509.pdf (624.9Kb)
    Access Status
    Open access
    Authors
    Ramsay, Joshua
    Date
    2013
    Type
    Journal Article
    
    Metadata
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    Citation
    Ramsay, J. 2013. High-throughput ß-galactosidase and ß-glucuronidase Assays Using Fluorogenic Substrates. Bio-protocol. 3 (14): Article e827.
    Source Title
    Bio-protocol
    DOI
    10.21769/BioProtoc.827
    School
    School of Biomedical Sciences
    URI
    http://hdl.handle.net/20.500.11937/46320
    Collection
    • Curtin Research Publications
    Abstract

    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.

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