Mahoney C W, Hosoi T, Ohashi M
Discovery Research Laboratory, Tanabe Pharmaceutical Co., Toda, Saitama, 335, Japan.
Anal Biochem. 1999 Mar 15;268(2):371-6. doi: 10.1006/abio.1998.3062.
Determination of phosphorylation sites in proteins is usually accomplished using [gamma-32P]ATP. For low-abundance phosphoproteins, in vivo and intact cell studies usually require millicurie levels of 32P for a single experiment, making multiple experiments prohibitive. Here we describe a low picomole sensitivity, nonradioisotopic, high-throughput method for tracing phosphorylation sites in proteins and peptides. The method is based on in situ enzyme-linked immunosorbent assay (ELISA) plate biotinylation of nonphospho- and phosphopeptides, streptavidin capture, and ELISA detection using recently available anti-phospho-Thr and anti-phospho-Ser antibodies.
蛋白质磷酸化位点的测定通常使用[γ-32P]ATP来完成。对于低丰度磷酸化蛋白质，体内和完整细胞研究通常在单次实验中需要毫居里水平的32P，这使得进行多次实验变得代价高昂。在此，我们描述了一种用于追踪蛋白质和肽段中磷酸化位点的低皮摩尔灵敏度、非放射性、高通量方法。该方法基于非磷酸化和磷酸化肽段的原位酶联免疫吸附测定（ELISA）板生物素化、链霉亲和素捕获以及使用最近可得的抗磷酸化苏氨酸和抗磷酸化丝氨酸抗体进行ELISA检测。
