Detection of protease activities using specific aminoacyl or peptidyl p- nitroanilides after sodium dodecyl sulfate - polyacrylamide gel electrophoresis and its applications

Wen Chi Hou, Hsien Jung Chen, Tzeng E. Chen, Yaw Huei Lin

Research output: Contribution to journalArticle

Abstract

A general method for detecting protease activities on acrylamide or agarose gels after sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) using specific aminoacyl p-nitroanilide (NA) or peptidyl NA as substrate is described. This method is extended from the spectrophotometric assay of p-nitroaniline, which is a chromogenic product liberated by protease action on aminoacyl NA or peptidyl NA. The acrylamide gel containing protein bands was dipped directly into a solution which contained specific synthetic aminoacyl NA or peptidyl NA as a substrate or had been overlaid with an agarose gel containing the same substrate. The p- nitroaniline released on the acrylamide or agarose get by the specific protease was diazotized with sodium nitrite and then coupled to N-(1- naphthyl)-ethylenediamine to produce distinct activity band(s). The substrates used for protease activity staining on gels were identical to those used for spectrophotometric assays. Some applications are described.

Original languageEnglish
Pages (from-to)486-490
Number of pages5
JournalElectrophoresis
Volume20
Issue number3
Publication statusPublished - 1999
Externally publishedYes

Keywords

  • Activity staining
  • p-Nitroanilide
  • Protease
  • Sodium dodecyl sulfate - polyacrylamide gel electrophoresis

ASJC Scopus subject areas

  • Clinical Biochemistry

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