An ultrasensitive, continuous fluorometric assay for calpain activity

Pétér Tompa, Éva Schád, Andrea Baki, Anita Alexa, József Batke, Peter Friedrich

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

A rapid, continuous assay for calcium-activated neutral protease activity is described. This assay is based on monitoring the elevation in fluorescence intensity that occurs upon calpainolytic digestion of dichlorotriazinylamino-fluorescein-labeled microtubule-associated protein 2. Tedious separation of peptide products from the protein substrate in this rapid assay is unnecessary, which thus offers two remarkable advantages over conventional caseinolytic assay procedures: (i) it raises sensitivity of detection by about three orders of magnitude, allowing the quantitative determination of calpain in the high picogram range in 10 min; and (ii) it permits a continuous detection of activity, which may prove invaluable in enzyme-mechanism studies that require pre-steady-state measurements. Other features and advantages of the assay, along with its limitations, are discussed in detail.

Original languageEnglish
Pages (from-to)287-293
Number of pages7
JournalAnalytical Biochemistry
Volume228
Issue number2
DOIs
Publication statusPublished - Jul 1995

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ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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