Spry Christina, Saliba Kevin J, Strauss Erick
Research School of Biology, College of Medicine, Biology and Environment, The Australian National University, Canberra, ACT 0200, Australia.
Research School of Biology, College of Medicine, Biology and Environment, The Australian National University, Canberra, ACT 0200, Australia; Medical School, College of Medicine, Biology and Environment, The Australian National University, Canberra, ACT 0200, Australia.
Anal Biochem. 2014 Apr 15;451:76-8. doi: 10.1016/j.ab.2013.12.010. Epub 2013 Dec 12.
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.
我们在此描述一种简单的、基于辐射的小型磷酸化分析方法，该方法可用于在存在纯化的和粗制酶制剂的情况下监测多种小分子底物的磷酸化。氢氧化钡（Ba(OH)₂）和硫酸锌（ZnSO₄）用于终止磷酰基转移，并在一步中选择性沉淀磷酸化反应产物；在定量之前，通过过滤除去未磷酸化的底物。与其他基于辐射的分析方法相比，其关键优势在于：（i）不需要高能/短寿命放射性发射体；（ii）无需高放射性浓度即可获得高质量数据；（iii）该分析方法与高通量应用兼容。
