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Titolo:
A simplified method for large scale quantification of transcriptional activity and its use in studies of steroids and steroid receptors
Autore:
Zhang, SM; Lu, JM; Iyama, K; Lo, SC; Danielsen, M;
Indirizzi:
Amer Registry Pathol, Washington, DC 20306 USA Amer Registry Pathol Washington DC USA 20306 ol, Washington, DC 20306 USA Georgetown Univ, Med Ctr, Dept Biochem & Mol Biol, Washington, DC 20007 USA Georgetown Univ Washington DC USA 20007 ol Biol, Washington, DC 20007 USA Armed Forces Inst Pathol, Washington, DC 20306 USA Armed Forces Inst Pathol Washington DC USA 20306 Washington, DC 20306 USA
Titolo Testata:
JOURNAL OF RECEPTOR AND SIGNAL TRANSDUCTION RESEARCH
fascicolo: 1, volume: 21, anno: 2001,
pagine: 71 - 84
SICI:
1079-9893(200102)21:1<71:ASMFLS>2.0.ZU;2-7
Fonte:
ISI
Lingua:
ENG
Soggetto:
CHLORAMPHENICOL ACETYLTRANSFERASE GENE; GLUCOCORTICOID RECEPTOR; WILD-TYPE; EXPRESSION; CELLS; DNA; ACTIVATION; DOMAINS; BINDING; ASSAY;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
17
Recensione:
Indirizzi per estratti:
Indirizzo: Zhang, SM Amer Registry Pathol, Washington, DC 20306 USA Amer Registry Pathol Washington DC USA 20306 gton, DC 20306 USA
Citazione:
S.M. 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

Abstract

Chloramphenicol acetyltransferase (CAT) is widely used as a reporter to determine the transcriptional specificity of promoters and for the quantification of transcriptional activity of transcription factors such as nuclear receptors. However, large-scale quantification of CAT activity in transfected mammalian cells is still heavily labor-intensive, time-consuming and expensive. Here, we describe a simplified method that combined using multiwell tissue culture plates in transfection and sample preparation and a modifiedsingle step method for quantitatively assaying CAT activity. By using multiwell plates, the tedious sample preparation procedure was dramatically simplified. The CAT assay is performed by mixing cell lysate, chloramphenicol,H-3-acetyl coenzyme A and non-aqueous scintillation fluid in scintillationvials, followed by automatically continuously counting samples two or three cycles at fixed time intervals. The catalytic reaction and determination of CAT activity are carried out in the vials simultaneously. This simplified protocol is faster, less expensive and more accurate than other CAT assayprocedures and the results can be normalized easily. The utility of the assay is demonstrated by the analysis of the transcriptional activity of the glucocorticoid and androgen receptors cotransfected into cells with a CAT reporter.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 18/01/20 alle ore 21:46:15