BMB Reports

Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
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Nano-scale Proteomics Approach Using Two-dimensional Fibrin Zymography Combined with Fluorescent SYPRO Ruby Dye

  • Choi, Nack-Shick (Proteome Research Laboratory, Korea Research Institute of Bioscience and Biotechnology) ;
  • Yoo, Ki-Hyun (Proteome Research Laboratory, Korea Research Institute of Bioscience and Biotechnology) ;
  • Yoon, Kab-Seog (Proteome Research Laboratory, Korea Research Institute of Bioscience and Biotechnology) ;
  • Maeng, Pil-Jae (Department of Microbiology, Chungnam National University) ;
  • Kim, Seung-Ho (Proteome Research Laboratory, Korea Research Institute of Bioscience and Biotechnology)
  • Published : 2004.05.31
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Abstract

In general, a SYPRO Ruby dye is well known as a sensitive fluorescence-based method for detecting proteins by one-or two-dimensional SDS-PAGE (1-DE or 2-DE). Based on the SYPRO Ruby dye system, the combined two-dimensional fibrin zymography (2-D FZ) with SYPRO Ruby staining was newly developed to identify the Bacillus sp. proteases. Namely, complex protein mixtures from Bacillus sp. DJ-4, which were screened from Doen-Jang (Korean traditional fermented food), showed activity on the zymogram gel. The gel spots on the SYPRO Ruby gel, which corresponded to the active spots showing on the 2-D FZ gel, were analyzed by a matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometric analysis. Five intracellular fibrinolytic enzymes of Bacillus sp. DJ-4 were detected through 2-D FZ. The gel spots on the SYPRO Ruby dye stained 2-D gel corresponding to 2-D FZ were then analyzed by MALID TOF MS. Three of the five gel spots proved to be quite similar to the ATP-dependent protease, extracellular neutral metalloprotease, and protease of Bacillus subtilis. Also, the extracellular proteases of Bacillus sp. DJ-4 employing this combined system were identified on three gels (e.g., casein, fibrin, and gelatin) and the proteolytic maps were established. This combined system of 2-D zymography and SYPRO Ruby dye should be useful for searching the specific protease from complex protein mixtures of many other sources (e.g., yeast and cancer cell lines).

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References

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