Diazo Coupling Method for Covalent Attachment of Proteins to Solid Substrates

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• 作者：Yang Wu ; Tione Buranda ; Robert L. Metzenberg ; Larry A. Sklar ; and Gabriel P. Lopez
• 刊名：Bioconjugate Chemistry
• 出版年：2006
• 出版时间：March 2006
• 年：2006
• 卷：17
• 期：2
• 页码：359 - 365
• 全文大小：220K
• 年卷期：v.17,no.2(March 2006)
• ISSN：1520-4812

文摘

We describe a process for covalently linking proteins to glass microscope slides and microbeads in a manner thatoptimizes the reactivity of the immobilized proteins and that is suitable for high-throughput microarray and flowcytometry analysis. The method involves the diazo coupling of proteins onto activated self-assembled monolayersformed from p-aminophenyl trimethoxysilane. Proteins immobilized by this method maintained bioactivity andproduced enhanced levels of protein-protein interaction, low background fluorescence, and high selectivity. Thebinding of immobilized proteins to their specific binding partner was analyzed quantitatively and successfullycorrelated with solution concentrations. Diazotized surfaces bound more efficiently to proteins containing ahexahistidine tag than those without a his-tag. Moreover, significantly higher reactivity of the immobilized his-tagged proteins was observed on diazotized surfaces than on amine-terminated surfaces. Results suggest thathis-tagged proteins are immobilized by reaction of the his-tag with the diazotized surface, thus offering the possibilityfor preferential orientation of covalently bound proteins.