A fast and robust procedure for the quantification of GFP-tagged membrane proteins in cell homogenates was developed employing capillary gel electrophoresis coupled to laser-induced fluorescence detection (CGE-LIF). The new method was found to be highly sensitive and applicable to structurally diverse membrane proteins including synaptic vesicle protein 2A (SV2A), adenosine A_2A receptor (A_2AAR), and connexin 43 (Cx43). Quantification of SV2A and A_2AAR using radioligand binding assays confirmed the results obtained with CGE-LIF. The CGE-LIF method showed significantly higher sensitivity as compared to fluorimetric measurement in a microplate. Importantly, CGE-LIF involves separation of the target proteins and their degradation products prior to quantification and thereby ensures specificity. We anticipate broad applicability of the method for any fluorophore-tagged protein.

中文翻译：

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毛细管凝胶电泳定量绿色荧光蛋白-（GFP-）标记的膜蛋白

利用毛细管凝胶电泳与激光诱导的荧光检测（CGE-LIF）结合，开发了一种快速，稳定的定量细胞匀浆中带GFP标签的膜蛋白的方法。发现该新方法高度灵敏，适用于结构多样的膜蛋白，包括突触小泡蛋白2A（SV2A），腺苷A _2A受体（A _2A AR）和连接蛋白43（Cx43）。SV2A和A _2A的定量使用放射性配体结合测定的AR证实了用CGE-LIF获得的结果。与微量培养板中的荧光测定法相比，CGE-LIF方法显示出显着更高的灵敏度。重要的是，CGE-LIF涉及在定量之前分离目标蛋白及其降解产物，从而确保了特异性。我们预期该方法对任何带有荧光团标记的蛋白质都具有广泛的适用性。