Buranda T, Lopez G P, Simons P, Pastuszyn A, Sklar L A
Department of Pathology, University of New Mexico School of Medicine, Albuquerque 87131, USA.
Anal Biochem. 2001 Nov 15;298(2):151-62. doi: 10.1006/abio.2001.5363.
Epitope tagging of expressed proteins is a versatile tool for the detection and purification of the proteins. This approach has been used in protein-protein interaction studies, protein localization, and immunoprecipitation. Among the most popular tag systems is the FLAG epitope tag, which is recognized by three monoclonal antibodies M1, M2, and M5. We describe novel approaches to the detection of epitope-tagged proteins via fluorescence resonance energy transfer on beads. We have synthesized and characterized biotinylated and fluorescein-labeled FLAG peptides and examined the binding of FLAG peptides to commercial streptavidin beads using flow cytometric analysis. A requirement of assay development is the elucidation of parameters that characterize the binding interactions between component systems. We have thus compiled a set of Kd values determined from a series of equilibrium binding experiments with beads, peptides, and antibodies. We have defined conditions for binding biotinylated and fluoresceinated FLAG peptides to beads. Site occupancies of the peptides were determined to be on the order of several million sites per bead and Kd values in the 0.3-2.0 nM range. The affinity for antibody attachment to peptides was determined to be in the low nanomolar range (less than 10 nM) for measurements on beads and solution. We demonstrate the applicability of this methodology to assay development, by detecting femtomole amounts of N-terminal FLAG-bacteria alkaline phosphatase fusion protein. These characterizations form the basis of generalizable and high throughput assays for proteins with known epitopes, for research, proteomic, or clinical applications.
对表达的蛋白质进行表位标记是检测和纯化蛋白质的一种通用工具。这种方法已用于蛋白质-蛋白质相互作用研究、蛋白质定位和免疫沉淀。最流行的标签系统之一是FLAG表位标签，它可被三种单克隆抗体M1、M2和M5识别。我们描述了通过珠子上的荧光共振能量转移检测表位标记蛋白质的新方法。我们合成并表征了生物素化和荧光素标记的FLAG肽，并使用流式细胞术分析检测了FLAG肽与商业链霉亲和素珠子的结合。分析方法开发的一个要求是阐明表征组分系统之间结合相互作用的参数。因此，我们汇总了一系列由珠子、肽和抗体的平衡结合实验确定的Kd值。我们定义了生物素化和荧光素化的FLAG肽与珠子结合的条件。确定肽的位点占有率约为每个珠子数百万个位点，Kd值在0.3-2.0 nM范围内。对于珠子和溶液中的测量，确定抗体与肽结合的亲和力处于低纳摩尔范围（小于10 nM）。我们通过检测飞摩尔量的N端FLAG-细菌碱性磷酸酶融合蛋白，证明了该方法在分析方法开发中的适用性。这些表征为具有已知表位的蛋白质的通用和高通量分析奠定了基础，可用于研究、蛋白质组学或临床应用。
