Development of gel staining techniques for detecting the secretion of procathepsin D (52-kDa protein) in MCF-7 human breast cancer cells

Venkatesh Krishnan, Tumkur Narasimhan, Stephen Safe

Research output: Contribution to journalArticle

26 Scopus citations

Abstract

17β-Estradiol stimulates the secretion of the 34- and 52-kDa protein (i.e., cathepsin D and procathepsin D, respectively) from MCF-7 human breast cancer cells and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) inhibits this estrogen-stimulated response. A comparison of the effects of 17β-estradiol, TCDD, and their combinations on the secretion of these two proteins was determined using four different assay procedures, namely autoradiographic analysis of the 35S-labeled proteins (from [35S]methionine) separated by polyacrylamide gel electrophoresis (PAGE), densitometric analysis of the silver- and double-stained proteins separated by PAGE, and radioimmunoassay of the proteins using commercially available antibodies to the 52-kDa protein. The results showed that the autoradiographic, staining, and radioimmunoassay procedures gave comparable results with only a few minor differences in the relative amounts of the 52-kDa detected in the various treatment groups. In the medium obtained from 17β-estradiol-treated cells that was serially diluted, there was an excellent linear correlation for the relative concentrations of the 52-kDa protein using the double-staining/densitometric procedure and the radioimmunoassay. These results indicate that the double- or silver-staining method may be a useful and rapid method for screening new compounds as antiestrogens in MCF-7 cells.

Original languageEnglish (US)
Pages (from-to)137-142
Number of pages6
JournalAnalytical Biochemistry
Volume204
Issue number1
DOIs
StatePublished - Jul 1992

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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