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CATA

Sigma-Aldrich

Chloramphenicol Acetyltransferase (CAT) Reporter Gene Activity Detection Kit

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About This Item

UNSPSC Code:
12352200
Pricing and availability is not currently available.

usage

 kit sufficient for 100 tests

shipped in

dry ice

storage temp.

−20°C

Application

One of the most widely used reporter genes in mammalian expression systems is chloramphenicol acetyltransferase (CAT) from Escherichia coli. Mammalian cells have minimal CAT activity, resulting in high signal-to-background ratios for this reporter gene assay. Conventional CAT assays use a radioactive substrate and measure acetylated chloramphenicol. This carries all the disadvantages of radioactive assays plus another that is peculiar to the CAT reaction. Chloramphenicol has two free hydroxyl groups, but only the 3-OH is acetylated by CAT. The 3-acetate can rearrange to the 1-position non-enzymatically, and to the extent that it does, the 3-OH is open for further acetylation. The result is that three products are produced: two monoacetates and a diacetate. In addition, because the transacetylation is slow, the quantity of products is not a true measure of the CAT activity.

Features and Benefits

  • Provides a non-radioactive method that does not suffer from the disadvantages of the conventional assay
  • The substrate, a fluorescent derivative of 1-deoxychloramphenicol, has only a 3-hydroxyl group; only one enzymatic product is possible
  • Substrate and product are separated cleanly on TLC; UV illumination of the developed plate offers a quick qualitative indication of CAT activity
  • Product and substrate can be extracted from the TLC plate for a quantitative fluorimetric assay.

Legal Information

The BODIPY-labeled substrate and product are covered by U.S. Patent Nos. 5,262,545 and 5,364,764.

Kit Components Only

Product No.
Description
  • Detailed technical bulletin 1 ea

Kit Components Also Available Separately

Product No.
Description
SDS
  • A2181Acetyl-CoA (cofactor) 1 mL/vialSDS
  • BODIPY FL–1-deoxychloramphenicol 3-acetate (positive standard) 1 mL/vial
  • BODIPY FL–1-deoxychloramphenicol (substrate) 1 mL/vial
  • C8413CAT enzyme (positive control) 250 USDS
  • 5× lysis buffer 25 mL

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Certificates of Analysis (COA)

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Uses of Fusion Genes in Mammalian Transfection.
Kain, S.R. & Ganguly, S., et al. et al.
Current Protocols in Molecular Biology, 1, 9-9 (1996)
C K Lefevre et al.
BioTechniques, 19(3), 488-493 (1995-09-01)
We report the development of fluorescent BODIPY 1-deoxychloramphenicol substrates for chloramphenicol acetyltransferase (CAT). These substrates not only simplify and improve quantitation of CAT activity but also extend the linear range of detection. Because the 1-deoxychloramphenicol derivatives have only one acetylation
Reporter genes: application to the study of mammalian gene transcription.
J Alam et al.
Analytical biochemistry, 188(2), 245-254 (1990-08-01)

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