Angeles T S, Steffler C, Bartlett B A, Hudkins R L, Stephens R M, Kaplan D R, Dionne C A
Department of Cell Biology, Cephalon, Inc., 145 Brandywine Parkway, West Chester, Pennsylvania, 19380, USA.
Anal Biochem. 1996 Apr 5;236(1):49-55. doi: 10.1006/abio.1996.0130.
A 96-well microtiter enzyme-linked immunosorbent assay was developed to assay the activity of the cytoplasmic domain of trkA tyrosine kinase. The assay involves immobilization of phospholipase C- gamma/glutathione S-transferase fusion protein on a microtiter plate, addition of the kinase reaction mixture, and detection by an antibody to phosphotyrosine followed by an alkaline phosphatase-conjugated second antibody. The substrate used in this system, phospholipase C-gamma, is one of several biologically important substrates for the phosphorylation reaction of receptor-linked tyrosine kinases. The assay was then used to characterize kinase inhibitory activities of various small molecules including analogs of K-252a.
开发了一种96孔微量滴定酶联免疫吸附测定法来检测trkA酪氨酸激酶胞质结构域的活性。该测定法包括将磷脂酶C-γ/谷胱甘肽S-转移酶融合蛋白固定在微量滴定板上，加入激酶反应混合物，并通过抗磷酸酪氨酸抗体随后是碱性磷酸酶偶联的二抗进行检测。该系统中使用的底物磷脂酶C-γ是受体连接的酪氨酸激酶磷酸化反应的几种生物学重要底物之一。然后该测定法用于表征包括K-252a类似物在内的各种小分子的激酶抑制活性。
