Detection of protease activities using specific aminoacyl or peptidyl p-nitroanilides after sodium dodecyl sulfate - polyacrylamide gel electrophoresis and its applications.
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.
Hou
W C
WC
Institute of Botany, Academica Sinica, Nankang, Taipei, Taiwan, ROC.
Chen
H J
HJ
Chen
T E
TE
Lin
Y H
YH
D001586:Benzoylarginine Nitroanilide / Q000378:metabolism; D002918:Chymotrypsin / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D010450:Endopeptidases / Q000378:metabolism*; D012967:Sodium Dodecyl Sulfate; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism
D012967:Sodium Dodecyl Sulfate; D001586:Benzoylarginine Nitroanilide; D010450:Endopeptidases; D002918:Chymotrypsin; D014357:Trypsin