A miniaturized assay for measuring small molecule phosphorylation in the presence of complex matrices

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• 作者：Christina Spry^a ; ¹ ; ^{christina.spry@anu.edu.au" class="auth_mail} ; Kevin J. Saliba^a ; ^b ; Erick Strauss^c ; ^{estrauss@sun.ac.za" class="auth_mail}
• 关键词：Phosphorylation assay ; Kinase ; Somogyi ; Pantothenate kinase ; Choline kinase ; Hexokinase
• 刊名：Analytical Biochemistry
• 出版年：15 April, 2014
• 年：2014
• 卷：451
• 期：Complete
• 页码：76-78
• 全文大小：527 K

文摘

We describe here a simple, miniaturized radiation-based phosphorylation assay that can be used to monitor phosphorylation of a diverse range of small molecule substrates in the presence of purified and crude enzyme preparations. Ba(OH)₂ and ZnSO₄ are used to terminate phosphoryl transfer and to precipitate selectively the phosphorylated reaction product in a single step; non-phosphorylated substrate is removed by filtration prior to quantification. The key advantages over alternative radiation-based assays are that: (i) high-energy/short-lived radioactive emitters are not required; (ii) high-quality data can be obtained without the need for high radioactivity concentrations; and (iii) the assay is compatible with high-throughput applications.