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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Abbreviations
- 4-MUG:
-
4-methylumbelliferyl-β-d-glucuronide
- BSA:
-
bovine serum albumin
- F:
-
fluorescence
- GUS:
-
β-glucuronidase
- uidA (synonym=gusA):
-
gene encoding GUS
- U:
-
unit
References
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Breyne, P., De Loose, M., Dedonder, A. et al. Quantitative kinetic analysis of β-glucuronidase activities using a computer-directed microtiter plate reader. Plant Mol Biol Rep 11, 21–31 (1993). https://doi.org/10.1007/BF02670556
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DOI: https://doi.org/10.1007/BF02670556