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An improved fluor diffusion assay for chloramphenicol acetyltransferase gene expression
1Med. Universität zu Lübeck, Institut für Med. Molekularbiologie, FRG.
Biotechniques
|February 1, 1994
Summary
We enhanced the fluor diffusion assay for chloramphenicol acetyltransferase (CAT) activity using [3H]acetyl CoA. This improved assay is more sensitive, has a wider linear range, and is less costly.
Area of Science:
- Biochemistry
- Enzyme Assays
Background:
- Chloramphenicol acetyltransferase (CAT) is widely used in molecular biology.
- Existing fluor diffusion assays for CAT activity have limitations in sensitivity and linearity.
Purpose of the Study:
- To significantly improve the fluor diffusion assay for chloramphenicol acetyltransferase (CAT) activity.
- To develop a more sensitive, linear, and cost-effective assay for CAT detection.
Main Methods:
- Utilized stable and inexpensive [3H]NaAcetate and yeast acetyl CoA synthetase to produce [3H]acetyl CoA with high specific radioactivity and yield.
- Introduced enzymatically produced [3H]acetyl CoA as a substrate in the fluor diffusion assay for CAT.
Main Results:
- Achieved substantial improvement in the fluor diffusion assay for CAT activity.
- The modified assay demonstrated increased sensitivity.
- The range of linearity was expanded by two orders of magnitude.
- The assay became less costly to perform.
Conclusions:
- The enhanced fluor diffusion assay offers superior performance for measuring CAT activity.
- This improved method provides a more sensitive, linear, and economical tool for researchers.
- The use of enzymatically produced [3H]acetyl CoA is key to the assay's advancements.