A method for increasing the sensitivity of chloramphenicol acetyltransferase assays in extracts of transfected cultured cells

David W. Crabb, Jack E. Dixon

Research output: Contribution to journalArticle

130 Scopus citations

Abstract

Transfection of several cell lines (HeLa, COS, PC-12, CA-77, and H4IIE C3) with pRSV-CAT by a variety of methods yielded rather low chloramphenicol acetyltransferase (CAT) activity in cell extracts. Extracts of these cells were found to interfere with the assay of added CAT. The extracts were capable of deacetylating acetylchloramphenicol and of accelerating the rate of hydrolysis of the acetyl-CoA present in the assay. Heating the cell extract to 60°C for 10 min completely prevented the interference and slowed the hydrolysis of acetyl-CoA. Substantially higher CAT activities were observed when the extract was heat treated in the presence of EDTA prior to enzyme assay for most cell lines tested. This simple reliable method makes possible the accurate assessment of CAT activities in different cell lines. These observations are particularly pertinent to investigators studying tissue-specific gene expression.

Original languageEnglish (US)
Pages (from-to)88-92
Number of pages5
JournalAnalytical biochemistry
Volume163
Issue number1
DOIs
StatePublished - May 15 1987

Keywords

  • Chloramphenicol acetyltransferase
  • gene expression
  • gene regulation
  • recombinant technology
  • transfection
  • transient expression

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

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