A membrane capture assay for lipid kinase activity
A membrane capture assay for lipid kinase activity
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DOI：
10.1038/nprot.2007.361
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发表时间：
2007-01-01
期刊：
影响因子：
14.8
通讯作者：
Shokat, Kevan M.
中科院分区：
文献类型：
--
作者：
Knight, Zachary A.;Feldman, Morri E.;Shokat, Kevan M.
磷脂酰肌醇激酶如PI 3-激酶合成控制不同细胞过程的脂质第二信使。最近，这些酶已成为一类重要的药物靶点，并有显着的兴趣，发现新的脂质激酶抑制剂。我们在这里描述了一个高通量测定脂质激酶抑制剂IC 50值的程序。该测定利用磷酸肌醇而不是核苷酸（如ATP）不可逆地结合到硝酸纤维素膜的事实。因此，可以通过将粗反应点在硝酸纤维素膜上然后洗涤来分离来自激酶测定的放射性标记脂质。我们表明，不同的磷酸肌醇激酶可以使用这种方法进行测定，并概述了如何在96孔板中进行测定。我们还描述了一个MATLAB脚本，自动化的数据分析。整个过程需要3-4小时。
Phosphoinositide kinases such as PI3-kinase synthesize lipid second messengers that control diverse cellular processes. Recently, these enzymes have emerged as an important class of drug targets, and there is significant interest in discovering new lipid kinase inhibitors. We describe here a procedure for the high-throughput determination of lipid kinase inhibitor IC50 values. This assay exploits the fact that phosphoinositides, but not nucleotides such as ATP, bind irreversibly to nitrocellulose membranes. As a result, the radiolabeled lipids from a kinase assay can be isolated by spotting the crude reaction on a nitrocellulose membrane and then washing. We show that diverse phosphoinositide kinases can be assayed using this approach and outline how to perform the assay in 96-well plates. We also describe a MATLAB script that automates the data analysis. The complete procedure requires 3-4 h.