Medina-Kauwe L K, Leung V, Wu L, Kedes L
University of Southern California Keck School of Medicine, Los Angeles, USA.
Biotechniques. 2000 Sep;29(3):602-4, 606-8, 609. doi: 10.2144/00293rr03.
We have developed a simple scheme for characterizing ligand-receptor binding and post-binding activity on living cells. Our approach makes use of green fluorescent protein (GFP) as an auto-fluorescent tag to label protein ligands. We have constructed GFP-tagged ligands that can be expressed in bacteria as soluble fusion proteins. A cell-binding assay using fluorescence-activated cell sorting (FACS) demonstrates that GFP-tagged proteins retain their wild-type receptor-binding specificity. Using this assay, we measure ligand binding on unfixed cells and demonstrate receptor specificity using specific competitors. To determine the ability of receptor targets to internalize, we developed a second FACS-based assay to detect the rate and percentage of internalized ligand in living cells. Noninternalizing control ligands and fluorescence microscopy of treated cells confirm that our assay is reliable for determining receptor internalization activity.
我们开发了一种简单的方案，用于表征活细胞上的配体-受体结合及结合后活性。我们的方法利用绿色荧光蛋白（GFP）作为自发荧光标签来标记蛋白质配体。我们构建了可在细菌中作为可溶性融合蛋白表达的GFP标记配体。使用荧光激活细胞分选（FACS）的细胞结合试验表明，GFP标记的蛋白质保留了其野生型受体结合特异性。使用该试验，我们测量未固定细胞上的配体结合，并使用特异性竞争剂证明受体特异性。为了确定受体靶点的内化能力，我们开发了第二种基于FACS的试验，以检测活细胞中内化配体的速率和百分比。非内化对照配体和处理后细胞的荧光显微镜检查证实，我们的试验对于确定受体内化活性是可靠的。
