Quantitative kinetic analysis of β-glucuronidase activities using a computer-directed microtiter plate reader |
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| Authors: | Peter Breyne Marc De Loose Andrée Dedonder Marc Van Montagu Ann Depicker |
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| Institution: | (1) Laboratorium voor Genetica, Universiteit Cent, B-9000 Gent, Belgium;(2) Laboratorium voor Plantenfysiologie, Universiteit Gent, B-9000 Gent, Belgium;(3) Present address: Rijksstation voor Plantenveredeling, Burg. Van Gansberghelaan, 109, B-9820 Merelbeke-Lemberge, Belgium |
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| Abstract: | We have established a procedure for automated, kinetic analysis of β-glucuronidase (GUS) activities using a colorimetric or
fluorometric microtiter plate reader connected to a computer that directs the measurements and accesses the data. Compared
with end-point measurements, the procedure saves time, is more accurate, and needs 20 times less material. It allows a more
precise determination of GUS activities over a range of 400,000-fold, with a limit of detection of about 0.01 units of GUS
per mL in the colorimetric assay and 0.1 milliunit of GUS in the fluorometric assay. A general protocol for the determination
of GUS activities in transgenic plant tissue was worked out and applied to investigate the expression of a chimeric β-glucuronidase
gene in stably transformed tobacco calli. |
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| Keywords: | GUS assay gene fusion reporter gene tobacco Nicotiana tabacum |
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