27
Views
2
CrossRef citations to date
0
Altmetric
Research Article

A SIMPLIFIED METHOD FOR LARGE SCALE QUANTIFICATION OF TRANSCRIPTIONAL ACTIVITY AND ITS USE IN STUDIES OF STEROIDS AND STEROID RECEPTORS

, , , &
Pages 71-84 | Published online: 28 Feb 2001
 

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 modified single 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, 3H-acetyl co-enzyme 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 of 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 assay is demonstrated by the analysis of the transcriptional activity of the glucocorticoid and androgen receptors cotransfected into cells with a CAT reporter.

Log in via your institution

Log in to Taylor & Francis Online

PDF download + Online access

  • 48 hours access to article PDF & online version
  • Article PDF can be downloaded
  • Article PDF can be printed
USD 65.00 Add to cart

Issue Purchase

  • 30 days online access to complete issue
  • Article PDFs can be downloaded
  • Article PDFs can be printed
USD 1,339.00 Add to cart

* Local tax will be added as applicable

Related Research

People also read lists articles that other readers of this article have read.

Recommended articles lists articles that we recommend and is powered by our AI driven recommendation engine.

Cited by lists all citing articles based on Crossref citations.
Articles with the Crossref icon will open in a new tab.