Diced Electrophoresis Gel Assay for Screening Enzymes with Specified Activities

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• 作者：Toru Komatsu ; Kenjiro Hanaoka ; Alexander Adibekian ; Kentaro Yoshioka ; Takuya Terai ; Tasuku Ueno ; Mitsuyasu Kawaguchi ; Benjamin F. Cravatt ; Tetsuo Nagano
• 刊名：The Journal of the American Chemical Society
• 出版年：2013
• 出版时间：April 24, 2013
• 年：2013
• 卷：135
• 期：16
• 页码：6002-6005
• 全文大小：312K
• 年卷期：v.135,no.16(April 24, 2013)
• ISSN：1520-5126

文摘

We have established the diced electrophoresis gel (DEG) assay as a proteome-wide screening tool to identify enzymes with activities of interest using turnover-based fluorescent substrates. The method utilizes the combination of native polyacrylamide gel electrophoresis (PAGE) with a multiwell-plate-based fluorometric assay to find protein spots with the specified activity. By developing fluorescent substrates that mimic the structure of neutrophil chemoattractants, we could identify enzymes involved in metabolic inactivation of the chemoattractants.