An enzyme-linked immunosorbent assay for the Raf/MEK1/MAPK signaling cascade
An enzyme-linked immunosorbent assay for the Raf/MEK1/MAPK signaling cascade
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DOI：
10.1006/abio.2001.5151
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发表时间：
2001-07-01
影响因子：
2.9
通讯作者：
Kohler, C
中科院分区：
文献类型：
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作者：
Mallon, R;Feldberg, LR;Kohler, C
Ras-MAPK信号级联将来自生长因子受体和活化的Ras的促有丝分裂刺激传递到细胞核。不适当的Ras激活与大约30%的人类癌症相关。Ras-MAPK信号级联中的激酶组分是药物干预的有吸引力的靶点，因此，我们开发了一种高通量、非放射性的ELISA方法来监测Raf和MEK 1激酶活性。在该测定系统中，活化的Raf磷酸化并活化MEK 1，其进而磷酸化MAPK。特异性检测磷酸化MAPK（与非磷酸化MAPK）的抗体使酶联免疫吸附测定（ELISA）的发展成为可能。该测定检测Raf和/或MEK 1的抑制剂，并已用于筛选大量的随机化合物。具体如下：Raf/MEK 1/MAPK ELISA中的抑制靶标随后可以通过直接测量MEK 1的Raf磷酸化和MAPK的MEK 1磷酸化的二级测定来鉴定。(C)北京：科学出版社.
The Ras-MAPK signaling cascade transmits mitogenic stimuli from growth factor receptors and activated Ras to the cell nucleus. Inappropriate Ras activation is associated with similar to 30% of all human cancers. The kinase components of the Ras-MAPK signaling cascade are attractive targets for pharmaceutical intervention, Therefore, we have developed a high-throughput, nonradioactive ELISA method to monitor Raf and MEK1 kinase activity. In this assay system activated Raf phosphorylates and activates MEK1, which in turn phosphorylates MAPK. Antibodies that specifically detect phosphorylated MAPK (vs nonphosphorylated MAPK) made enzyme-linked immunosorbent assay (ELISA) development possible. This assay detects inhibitors of Raf and/or MEK1 and has been used to screen large numbers of random compounds. The specific: target of inhibition in the Raf/MEK1/MAPK ELISA can be subsequently identified by secondary assays which directly measure Raf phosphorylation of MEK1 car MEK1 phosphorylation of MAPK. (C) 2001 Academic Press.