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Detection of protease activities using specific aminoacyl or peptidyl p-nitroanilides after sodium dodecyl sulfate - polyacrylamide gel electrophoresis and its applications.
Electrophoresis. 1999 Mar; 20(3):486-90.E

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 gel 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.

Authors+Show Affiliations

Hou WC
Institute of Botany, Academica Sinica, Nankang, Taipei, Taiwan, ROC.
Chen HJ
No affiliation info available
Chen TE
No affiliation info available
Lin YH
No affiliation info available

MeSH

Benzoylarginine NitroanilideChymotrypsinElectrophoresis, Polyacrylamide GelEndopeptidasesSodium Dodecyl SulfateSubstrate SpecificityTrypsin

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

10217159

Citation

Hou, W C., et al. "Detection of Protease Activities Using Specific Aminoacyl or Peptidyl P-nitroanilides After Sodium Dodecyl Sulfate - Polyacrylamide Gel Electrophoresis and Its Applications." Electrophoresis, vol. 20, no. 3, 1999, pp. 486-90.
Hou WC, Chen HJ, Chen TE, et al. Detection of protease activities using specific aminoacyl or peptidyl p-nitroanilides after sodium dodecyl sulfate - polyacrylamide gel electrophoresis and its applications. Electrophoresis. 1999;20(3):486-90.
Hou, W. C., Chen, H. J., Chen, T. E., & Lin, Y. H. (1999). Detection of protease activities using specific aminoacyl or peptidyl p-nitroanilides after sodium dodecyl sulfate - polyacrylamide gel electrophoresis and its applications. Electrophoresis, 20(3), 486-90.
Hou WC, et al. Detection of Protease Activities Using Specific Aminoacyl or Peptidyl P-nitroanilides After Sodium Dodecyl Sulfate - Polyacrylamide Gel Electrophoresis and Its Applications. Electrophoresis. 1999;20(3):486-90. PubMed PMID: 10217159.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Detection of protease activities using specific aminoacyl or peptidyl p-nitroanilides after sodium dodecyl sulfate - polyacrylamide gel electrophoresis and its applications. AU - Hou,W C, AU - Chen,H J, AU - Chen,T E, AU - Lin,Y H, PY - 1999/4/27/pubmed PY - 2000/8/12/medline PY - 1999/4/27/entrez SP - 486 EP - 90 JF - Electrophoresis JO - Electrophoresis VL - 20 IS - 3 N2 - 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 gel 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. SN - 0173-0835 UR - https://www.unboundmedicine.com/medline/citation/10217159/Detection_of_protease_activities_using_specific_aminoacyl_or_peptidyl_p_nitroanilides_after_sodium_dodecyl_sulfate___polyacrylamide_gel_electrophoresis_and_its_applications_ L2 - https://doi.org/10.1002/(SICI)1522-2683(19990301)20:3<486::AID-ELPS486>3.0.CO;2-# DB - PRIME DP - Unbound Medicine ER -