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A nonradioactive assay for transfected chloramphenicol acetyltransferase activity using fluorescent substrates.

Publication ,  Journal Article
Young, SL; Barbera, L; Kaynard, AH; Haugland, RP; Kang, HC; Brinkley, M; Melner, MH
Published in: Anal Biochem
September 2, 1991

Studies of the transcriptional activity of gene promoters have been greatly assisted by the widespread use of the chloramphenicol acetyltransferase (CAT) gene as a reporter gene. Previous techniques for assaying CAT enzymatic activity have utilized radioactive substrates or cofactors with the resulting complications of handling radioactive materials. We report here the development of fluorescent substrates for the CAT enzyme which form the basis of a CAT enzyme assay of enzyme kinetic parameters (Km and Vmax) and sensitivity similar to those based on radioactive substrates. Fluorescent substrates were designed as analogs of chloramphenicol and were based on the structure-function requirements of the enzyme. Several fluorophores were used to derivatize chloramphenicol base; one of the most effective was the borondipyrromethene difluoride (BODIPY) fluorophore. One BODIPY-chloramphenicol analog was found to have a Km for the purified CAT enzyme of 2 microM (compared to 12 microM for 14C-labeled chloramphenicol) and a Vmax of 120 pmole/min (compared to 180 pmol/min for the radioactive substrate). To verify its usefulness, a BODIPY--chloramphenicol-based CAT assay was used to measure transient transfection of primary cultures of ovarian granulosa cells in serum-free medium. This experimental system requires a highly sensitive assay for detecting transfected CAT gene activity. Robust expression of CAT activity was easily detected in crude cellular extracts using FluoReporter FAST CAT, a kit containing the BODIPY-chloramphenicol analog. The expression was precisely quantified by methanol extraction of the substrate and products from TLC plates and subsequent measurement of fluorescence using excitation-emission spectroscopy.(ABSTRACT TRUNCATED AT 250 WORDS)

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Published In

Anal Biochem

DOI

ISSN

0003-2697

Publication Date

September 2, 1991

Volume

197

Issue

2

Start / End Page

401 / 407

Location

United States

Related Subject Headings

  • Transfection
  • Recombinant Fusion Proteins
  • Rats
  • Progesterone
  • Granulosa Cells
  • Gene Expression Regulation
  • Fluorescent Dyes
  • Fluorescence
  • Female
  • Chloramphenicol O-Acetyltransferase
 

Citation

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Young, S. L., Barbera, L., Kaynard, A. H., Haugland, R. P., Kang, H. C., Brinkley, M., & Melner, M. H. (1991). A nonradioactive assay for transfected chloramphenicol acetyltransferase activity using fluorescent substrates. Anal Biochem, 197(2), 401–407. https://doi.org/10.1016/0003-2697(91)90411-l
Young, S. L., L. Barbera, A. H. Kaynard, R. P. Haugland, H. C. Kang, M. Brinkley, and M. H. Melner. “A nonradioactive assay for transfected chloramphenicol acetyltransferase activity using fluorescent substrates.Anal Biochem 197, no. 2 (September 2, 1991): 401–7. https://doi.org/10.1016/0003-2697(91)90411-l.
Young SL, Barbera L, Kaynard AH, Haugland RP, Kang HC, Brinkley M, et al. A nonradioactive assay for transfected chloramphenicol acetyltransferase activity using fluorescent substrates. Anal Biochem. 1991 Sep 2;197(2):401–7.
Young, S. L., et al. “A nonradioactive assay for transfected chloramphenicol acetyltransferase activity using fluorescent substrates.Anal Biochem, vol. 197, no. 2, Sept. 1991, pp. 401–07. Pubmed, doi:10.1016/0003-2697(91)90411-l.
Young SL, Barbera L, Kaynard AH, Haugland RP, Kang HC, Brinkley M, Melner MH. A nonradioactive assay for transfected chloramphenicol acetyltransferase activity using fluorescent substrates. Anal Biochem. 1991 Sep 2;197(2):401–407.
Journal cover image

Published In

Anal Biochem

DOI

ISSN

0003-2697

Publication Date

September 2, 1991

Volume

197

Issue

2

Start / End Page

401 / 407

Location

United States

Related Subject Headings

  • Transfection
  • Recombinant Fusion Proteins
  • Rats
  • Progesterone
  • Granulosa Cells
  • Gene Expression Regulation
  • Fluorescent Dyes
  • Fluorescence
  • Female
  • Chloramphenicol O-Acetyltransferase