( loramphenicol. In the common CAT assay...)Transfection Reagent Selector Kit Handbook

loramphenicol. In the common CAT assay, cell lysates prepared from transfected cells are incubated with 14C-labeled chloramphenicol. The resulting acetylated and unacetylated forms of chloramphenicol are separated by thin-layer chromatography. A qualitative estimate of CAT activity can be obtained simply by exposing the plates to X-ray film. For quantitative analysis, the separated bands can be scraped from the thinlayer plate and the levels of radioactivity measured in a scintillation counter. Currently, a CAT ELISA is also often used. In this assay the total expression of the chloramphenicol acetyltransferase is measured via antibody detection, in contrast to the classic CAT assay described above, which determines only the active protein.

Firefly luciferase

Luciferase catalyses a bioluminescent reaction involving the substrate luciferin, ATP, Mg2+, and molecular oxygen. When these components are mixed with cell lysates containing luciferase, a flash of light is emitted. Light signals are detected using a luminometer or a liquid scintillation counter.

-Galactosidase

The prokaryotic enzyme b-galactosidase can be assayed colorimetrically using the substrate o-nitrophenyl-b-D-galactopyranoside (ONPG). The hydrolysis of ONPG by b-galactosidase yields a yellow-colored product, o-nitrophenol, which can be measured photometrically.

Human growth hormone (hGH)

The assay for human growth hormone is based on immunological detection of hGH secreted by transfected cells. Specific ¹²⁵I-labeled antibodies against hGH are used and results are monitored in a scint
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