CATA

Chloramphenicol Acetyltransferase (CAT) Reporter Gene Activity Detection Kit

• UNSPSC Code: 12352200

Eigenschaften

• usage: kit sufficient for 100 tests
• shipped in: dry ice
• storage temp.: −20°C

Beschreibung

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.

Nur Kit-Komponenten

• Detailed technical bulletin 1 ea

Kit-Komponenten auch einzeln erhältlich

• A2181

  Acetyl-CoA (cofactor) 1 mL/vial

  SDB
• BODIPY FL–1-deoxychloramphenicol 3-acetate (positive standard) 1 mL/vial

  SDB
• BODIPY FL–1-deoxychloramphenicol (substrate) 1 mL/vial

  SDB
• C8413

  CAT enzyme (positive control) 250 U

  SDB
• 5× lysis buffer 25 mL

  SDB