Nano-scale proteomics approach using two-dimensional fibrin zymography combined with fluorescent SYPRO Ruby dye

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Title
Nano-scale proteomics approach using two-dimensional fibrin zymography combined with fluorescent SYPRO Ruby dye
Author(s)
Nack Shick Choi; Ki Hyun Yoo; Kab Seog Yoon; P J Maeng; Seung Ho Kim
Bibliographic Citation
BMB Reports, vol. 37, no. 3, pp. 298-303
Publication Year
2004
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).
Keyword
proteomicsSYPRO rubytwo-dimensionbacilluszymography
ISSN
1225-8687
Publisher
Korea Soc-Assoc-Inst
DOI
http://dx.doi.org/10.5483/bmbrep.2004.37.3.298
Type
Article
Appears in Collections:
1. Journal Articles > Journal Articles
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