Chloramphenicol acetyltransferase (CAT) assays are a fast, easy and accurate method of measuring gene promoter activity
in vitro. As the CAT gene is not expressed in eukaryotic cells, by inserting a specific enhancer/promoter upstream of this gene in a plasmid, transfecting it into eukaryotic cells and modifying the cells with transcriptional enhancers (e.g. mitogens, transcription factors, drugs, serum, etc.), promoter activity can be measured by quantifying the amount of CAT protein made.
The Roche CAT ELISA Kit allows fast, accurate measurement of CAT in lysates from a transfected, CAT-expressing cell line, through the use of ELISA (enzyme-linked immuno-sorbent assay) microplates coated with anti-CAT antibody. In brief, anti-CAT antibodies are adsorbed onto the base of the microplate wells (each well is the same size as in a standard 96-well tissue culture plate). Lysate containing CAT is added to the plates and following a series of incubations and washes, the following are added to the ELISA plate: anti-CAT DIG (digoxigenin), anti-DIG POD (a peroxidase labeled anti-DIG antibody fragment) and finally Roche’s patented “ABTS” peroxidase substrate, which completes the colorimetric reaction. The CAT protein concentration can be determined using a standard plate reader, with absorbance measured at 405 nm. A control (CAT enzyme) which can be serially diluted to provide accurate concentration standards is also included with the kit.
The Roche CAT ELISA Kit provides several advantages over traditional CAT assays: There is no use of radioactive isotopes, a result is obtainable in approximately 5 hours (from start to finish), and the protocol and operating instructions are among the easiest of any kit I have used. Although ultimately the measure of the CAT protein is indirect, I find that an enormous advantage of this kit is that the reaction measures the actual amount of CAT protein, rather than CAT protein activity, which can degrade over time and is also dependent on the quality and stability of substrates.
I have used Roche’s CAT ELISA Kit to analyze the effect of transcription factor mutants on a CAT reporter plasmid. By transfecting my gene mutants into a cell line, along with the CAT reporter, I have measured the effects of these mutants on transcriptional activity. It is also important to measure transfection efficiency in order to know whether different levels of CAT are due to decreased/increased copies of plasmid getting into cells. This can be done in several ways – I usually co-transfect the cells with a plasmid encoding beta-galactosidase and measure activity through the addition of beta-galactosidase substrate (another colorimetric reaction), but other lab members have performed real-time quantitative PCR (qPCR) of the CAT plasmid itself in order to normalize transfection levels.
In summary, Roche’s CAT ELISA Kit provides a fast, reliable and easy-to-use system for measuring in vitro CAT protein levels. The instructions are clear and reasonably detailed; clear contact details are also provided for customer service. More information about the biology of CAT and applications of CAT assays may have been useful. Additionally, as the microplates are not sold separately, we always end up with left-over reagents, which is slightly wasteful. These are very minor inconveniences, however, and I cannot imagine a simpler or more informative method for conducting CAT assays.
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Sebastian Dworkin
Graduate Student
Research Department
Peter MacCallum Cancer Centre
Australia
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