CAT Enzyme Assay System

The CAT Enzyme Assay System offers two alternative methods for monitoring CAT enzyme activity in transfected cells: liquid scintillation counting (LSC) and thin layer chromatography (TLC). Either the LSC or TLC assays can be performed using the same cell extract. The TLC-based assay is less sensitive and more time-consuming to perform than the LSC assay but is useful as a visual confirmation of assay results. The resolved TLC reaction products are detected by autoradiography or phosphorimaging analysis.

Chloramphenicol Acetyltransferase (CAT), encoded by a bacterial drug-resistance gene, catalyzes the transfe...

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The CAT Enzyme Assay System offers two alternative methods for monitoring CAT enzyme activity in transfected cells: liquid scintillation counting (LSC) and thin layer chromatography (TLC). Either the LSC or TLC assays can be performed using the same cell extract. The TLC-based assay is less sensitive and more time-consuming to perform than the LSC assay but is useful as a visual confirmation of assay results. The resolved TLC reaction products are detected by autoradiography or phosphorimaging analysis.

Chloramphenicol Acetyltransferase (CAT), encoded by a bacterial drug-resistance gene, catalyzes the transfer of an acetyl group from acetyl-CoA to the 3´-hydroxy position of chloramphenicol. The enzyme is suitable as a standard in CAT assays of crude cell extracts. One unit is defined as the amount of enzyme required to transfer 1nmol of butyrate or acetate to chloramphenicol in one minute at 37°C.

n-Butyryl CoA is suitable for use in the chloramphenicol acetyltransferase (CAT) reaction. Transfer of the n-butyryl moiety to chloramphenicol by the CAT enzyme allows enzyme activity to be monitored using liquid scintillation counting or thin layer chromatography formats.

Features - Benefits

• Fast: The assay is performed in as little as 2–3 hours.
• Linear: The LSC assay is linear for three orders of magnitude of enzyme activity.
• Sensitive: As little as 3 × 10^–4 units (2pg) of CAT can be detected.
• Robust: Reporter Lysis Buffer allows luciferase, CAT and β-galactosidase assays to be performed from the same cell extract.

References

1. Shaw, W.V. (1975) Meth. Enzymol. 43, 737–55.
2. Gorman, C.M. et al. (1982) Mol. Cell. Biol. 2, 1044–51.
3. Seed, B. and Sheen, J.Y. (1988) Gene 67, 271–7.
4. Schenborn, E. and Titus, D. (1989) Promega Notes 21, 1–3.

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