Serine Protease Zymography: Low-Cost, Rapid, and Highly Sensitive RAMA Casein Zymography

Methods Mol Biol. 2017:1626:13-24. doi: 10.1007/978-1-4939-7111-4_2.

Abstract

To detect serine protease activity by zymography, casein and CBB stain have been used as a substrate and a detection procedure, respectively. Casein zymography has been using substrate concentration at 1 mg/mL and employing conventional CBB stain. Although ordinary casein zymography provides reproducible results, it has several disadvantages including time-consuming and relative low sensitivity. Improved casein zymography, RAMA casein zymography, is rapid and highly sensitive. RAMA casein zymography completes the detection process within 1 h after incubation and increases the sensitivity at least by tenfold. In addition to serine protease, the method also detects metalloprotease 7 (MMP7, Matrilysin) with high sensitivity.

Keywords: CBB stain; Casein zymography; Electrophoresis; MMP7; Proteinase; RAMA stain; SDS-PAGE; Serine protease.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Caseins / metabolism*
  • Coloring Agents / analysis
  • Coloring Agents / metabolism
  • Electrophoresis, Polyacrylamide Gel / economics
  • Electrophoresis, Polyacrylamide Gel / methods*
  • Enzyme Assays / methods*
  • Humans
  • Indicators and Reagents
  • Matrix Metalloproteinase 7 / analysis
  • Matrix Metalloproteinase 7 / metabolism
  • Rosaniline Dyes / analysis
  • Rosaniline Dyes / metabolism
  • Serine Proteases / analysis*
  • Serine Proteases / metabolism
  • Staining and Labeling / methods
  • Substrate Specificity
  • Time Factors

Substances

  • Caseins
  • Coloring Agents
  • Indicators and Reagents
  • Rosaniline Dyes
  • Coomassie brilliant blue R
  • Serine Proteases
  • Matrix Metalloproteinase 7