Histochemistry

, Volume 81, Issue 2, pp 167–169 | Cite as

Isoelectric focusing (IEF) and band detection with fluorogenic protease substrates

  • P. Sinha
  • R. Gossrau
Article

Summary

In a previous paper, combined dye histochemistry and analytical isoelectric focusing (IEF) of supernatants from organ homogenates have been shown to yield good results for the detection of protease isoenzymes. Difficulties arise when the protease to be studied is partially or completely inhibited y diazonium salts and when synthetic peptide substrates different from 4-methoxy-2-naphthylamine (MNA) amino acids and peptides are to be used. In this paper a technique is described in which cellulose acetate foils impregnated with MNA, 7-amino-4-methylcoumarin (AMC) and 7-amino-4-trifluoromethylcoumarin (AFC) substrates are overlaid on electrophoresis strips after IEF. After incubation, the foils are viewed with an UV-lamp and photographed. The MNA, AMC and AFC peptides are equally suitable for fluorescence band detection. Using this technique, occasionally protease isoenzymes are found which are more sensitive towards diazonium salts, e.g. aminopeptidase A and M. Sometimes it is also possible to detect thiolproteases which is not the case when employing dye histochemistry.

Keywords

Public Health Peptide Acetate Cellulose Cellulose Acetate 

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Copyright information

© Springer-Verlag 1984

Authors and Affiliations

  • P. Sinha
    • 1
  • R. Gossrau
    • 1
  1. 1.Department of AnatomyFree University of BerlinBerlin 33Federal Republic of Germany

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