Bioluminescence assay for detecting cell surface membrane protein expression.
Bioluminescence assay for detecting cell surface membrane protein expression.
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用于检测细胞表面膜蛋白表达的生物发光测定。
DOI：
10.1089/adt.2010.0278
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发表时间：
2011
影响因子：
1.8
通讯作者：
Hanyu,Yoshiro
中科院分区：
文献类型：
--
作者：
Kato,Mieko;Chiba,Tomoki;Li,Min;Hanyu,Yoshiro
我们已经开发了一种方法来测量细胞表面表达的膜蛋白的量与生物发光。甲藻荧光素酶在哺乳动物细胞表面上表达为与感兴趣的膜蛋白的嵌合融合蛋白。使用膜不可渗透的底物来定量膜展示的荧光素酶，测定细胞表面上的膜蛋白的表达。通过包括荧光素酶在细胞表面上的发光活性的猝灭步骤，我们能够通过测量猝灭后从细胞表面的发光恢复来监测膜蛋白表达动力学。报道的方法提供了一种方便的方法来监测表达和转运的膜蛋白到细胞表面的动力学。它适用于药物或候选药物对膜蛋白表达影响的高通量分析。
We have developed a method to measure the amounts of cell surface-expressed membrane proteins with bioluminescence. Dinoflagellate luciferase was expressed on the surface of a mammalian cell as a chimeric fusion protein with a membrane protein of interest. Using a membrane-impermeable substrate to quantify the membrane-displayed luciferase, the expression of the membrane protein on the cell surface was determined. By inclusion of a quenching step for the luminescent activity of luciferase on the cell surface, we were able to monitor the membrane protein expression kinetics by measuring the luminescence recovery from the cell surface after quenching. The reported methods provide a convenient way to monitor the kinetics of expression and transport of membrane proteins to the cell surface. It is applicable to the high-throughput analysis of drugs or drug candidates concerning their effects on membrane protein expression.