| Info. | Vol.6 - No.4 (2012.12.20) |
|---|---|
| Title | Characterization of TAMRA- and biotin-conjugated peptide arrays for on-chip matrix metalloproteinase activity assay |
| Authors | Deok-Hoon Kong1, Mahendra Prasad Bhatt1 Seung-Taek Lee2, Young-Myeong Kim1 & Kwon-Soo Ha1 |
| Institutions | 11Department of Molecular and Cellular Biochemistry and Institute of
Medical Science, Kangwon National University School of Medicine,
Chuncheon, Gangwon-Do 200-701, Korea 2Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, Korea Correspondence and requests for materials should be addressed to K.S. Ha ( ksha@kangwon.ac.kr) |
| Abstract | Peptide arrays have been widely used for high-throughput determination of protease activities in cells and tissues because specific peptides have high binding affinity for the active site of enzymes. Designing peptide substrate probes for enzyme activity assays have been considered to be important; however, the significance of its reporter tag for detecting enzymatic reactions is relatively underestimated. Thus, we investigated the effect of the reporter tag of peptide substrate probes on on-chip protease activity assays. We optimized and characterized proteolytic activity assay of matrix metalloproteinase-3 using direct and indirect substrate probes, tetramethyl-6-carboxyrhodamine (TAMRA)- and biotin-conjugated peptide arrays, respectively. Proteolytic activity assays using both substrate probes demonstrated similar sensitivity, ratio of maximal to minimal FI, IC50 of GM6001, and interarray reproducibility. However, biotin-conjugated substrate arrays showed a wider dynamic range than TAMRA-conjugated substrate arrays. Thus, this comparative study provides a wealth of information for developing optimal probes necessary for effective analysis of enzyme activity and kinetics. |
| Keyword | Peptide array, Matrix metalloproteinase-3, Substrate probes, TAMRA-conjugated peptide arrays, Biotin-conjugated peptide arrays |
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