An Ultrasensitive, Continuous Fluorometric Assay for Calpain Activity

P. Tompa, E. Schad, A. Baki, A. Alexa, J. Batke, P. 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

Fingerprint

Calpain
Assays
Microtubule-Associated Proteins
Fluorescein
Digestion
Fluorescence
Peptides
Enzymes
Proteins
Monitoring
Substrates

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology
  • Biophysics
  • Biochemistry

Cite this

An Ultrasensitive, Continuous Fluorometric Assay for Calpain Activity. / Tompa, P.; Schad, E.; Baki, A.; Alexa, A.; Batke, J.; Friedrich, P.

In: Analytical Biochemistry, Vol. 228, No. 2, 07.1995, p. 287-293.

Research output: Contribution to journalArticle

Tompa, P. ; Schad, E. ; Baki, A. ; Alexa, A. ; Batke, J. ; Friedrich, P. / An Ultrasensitive, Continuous Fluorometric Assay for Calpain Activity. In: Analytical Biochemistry. 1995 ; Vol. 228, No. 2. pp. 287-293.
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