Protein Microarray System for Detecting Protein-Protein Interactions Using an Anti-His-Tag Antibody and Fluorescence Scanning: Effects of the Heme Redox State on Protein-Protein Interactions of Heme-R

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• 作者：Yukie Sasakura ; Katsuhiro Kanda ; Tokiko Yoshimura-Suzuki ; Takuya Matsui ; Shinichi Fukuzono ; Moon Hi Han ; and Toru Shimizu
• 刊名：Analytical Chemistry
• 出版年：2004
• 出版时间：November 15, 2004
• 年：2004
• 卷：76
• 期：22
• 页码：6521 - 6527
• 全文大小：377K
• 年卷期：v.76,no.22(November 15, 2004)
• ISSN：1520-6882

文摘

A highly sensitive microarray system for detecting protein-protein interactions has been developed. This method wassuccessfully applied to analyze the interactions of heme-regulated phosphodiesterase from Escherichia coli (EcDOS). To immobilize (His)₆-Tag fused Ec DOS, anti-(His)₆-Tag monoclonal antibody (anti-(His)₆-Tag mAb) wasinitially immobilized on the solid surface, and (His)₆-Tagfused Ec DOS was fixed by antigen-antibody interactions.For this experiment, ProteoChip, generally suitable forantibody immobilization, was used as solid substrate. Inthis report, we confirm the antibody immobilization abilityof ProteoChip and specific binding to the F_(c) region ofthe antibody. Based on this finding, interdomain interactions between Ec DOS and the isolated heme-bound PASdomain were investigated on the solid surface. Ec DOSimmobilized via anti-(His)₆-Tag mAb maintained interactions with the PAS fragment, in contrast to directlyimmobilized Ec DOS in the absence of anti-(His)₆-TagmAb. Heme-redox-sensitive interactions between Ec DOSand the PAS fragment were additionally detected usinganti-(His)₆-Tag mAb as a mediator. Our results collectivelysuggest that the immobilization method using anti-Tagantibody is suitable for maintaining native protein characteristics to facilitate elucidation of their structures andfunctions on solid surfaces.