Sm. Zhang et al., A simplified method for large scale quantification of transcriptional activity and its use in studies of steroids and steroid receptors, J RECEPT SI, 21(1), 2001, pp. 71-84
Citations number
17
Categorie Soggetti
Cell & Developmental Biology
Journal title
JOURNAL OF RECEPTOR AND SIGNAL TRANSDUCTION RESEARCH
Chloramphenicol acetyltransferase (CAT) is widely used as a reporter to det
ermine the transcriptional specificity of promoters and for the quantificat
ion of transcriptional activity of transcription factors such as nuclear re
ceptors. However, large-scale quantification of CAT activity in transfected
mammalian cells is still heavily labor-intensive, time-consuming and expen
sive. Here, we describe a simplified method that combined using multiwell t
issue culture plates in transfection and sample preparation and a modified
single step method for quantitatively assaying CAT activity. By using multi
well plates, the tedious sample preparation procedure was dramatically simp
lified. The CAT assay is performed by mixing cell lysate, chloramphenicol,
H-3-acetyl coenzyme A and non-aqueous scintillation fluid in scintillation
vials, followed by automatically continuously counting samples two or three
cycles at fixed time intervals. The catalytic reaction and determination o
f CAT activity are carried out in the vials simultaneously. This simplified
protocol is faster, less expensive and more accurate than other CAT assay
procedures and the results can be normalized easily. The utility of the ass
ay is demonstrated by the analysis of the transcriptional activity of the g
lucocorticoid and androgen receptors cotransfected into cells with a CAT re
porter.