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Chloramphenicol acetyltransferase assay.

Stephen T Smale

Cold Spring Harbor Protocols

|May 5, 2010

Summary

This summary is machine-generated.

Quantifying promoter strength in transfection assays is crucial. This protocol uses chloramphenicol acetyltransferase (CAT) reporter gene assays to measure promoter activity by quantifying enzyme-catalyzed acetylation of a substrate.

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Area of Science:

Molecular Biology
Biochemistry
Genetics

Background:

Quantifying promoter strength is essential for gene expression studies.
Transfection efficiency can be low, necessitating sensitive reporter gene assays.
Reporter proteins provide a measurable readout of promoter activity.

Purpose of the Study:

To describe a protocol for quantifying promoter strength using a chloramphenicol acetyltransferase (CAT) reporter gene assay.
To detail the steps involved in cell lysis, enzyme reaction, product separation, and quantification.
To provide a reliable method for measuring promoter activity in transient and stable transfection assays.

Main Methods:

Cells are transfected with a CAT reporter plasmid.
Cell lysates are incubated with [(14)C]chloramphenicol and acetyl-coenzyme A for CAT-catalyzed acetylation.

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Acetylated products are separated from reactants via organic extraction and thin-layer chromatography (TLC).

Quantification is achieved through PhosphorImager analysis, scintillation counting, or densitometry.

Main Results:

The protocol enables sensitive measurement of CAT enzyme activity.
Percent conversion of substrate to product directly correlates with promoter strength.
Multiple quantification methods (TLC, direct extraction) are presented.

Conclusions:

The chloramphenicol acetyltransferase (CAT) assay is a robust method for quantifying promoter strength.
This protocol offers flexibility in quantification techniques for diverse laboratory settings.
Accurate promoter strength measurement is vital for understanding gene regulation.