Detection of epitope-tagged proteins in flow cytometry: Fluorescence resonance energy transfer-based assays on beads with femtomole resolution

Epitope tagging of expressed proteins is a versatile tool for the detection and purification of the proteins. This approach has been used in protein-p rotein interaction studies, protein localization, and immunoprecipitation. Among the most popular tag systems is the FLAG epitope tag, which is recogn ized by three monoclonal antibodies M1, M2, and M5. We describe novel appro aches to the detection of epitope-tagged proteins via fluorescence resonanc e energy transfer on beads. We have synthesized and characterized biotinyla ted 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 cha racterize 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 conditio ns for binding biotinylated and fluoresceinated FLAG peptides to beads. Sit e occupancies of the peptides were determined to be on the order of several million sites per bead and K-d values in the 0.3-2.0 nM range. The affinit y for antibody attachment to peptides was determined to be in the low nanom olar range (less than 10 nM) for measurements on beads and solution. We dem onstrate the applicability of this methodology to assay development, by det ecting femtomole amounts of N-terminal FLAG-bacteria alkaline phosphatase f usion protein. These characterizations form the basis of generalizable and high throughput assays for proteins with known epitopes, for research, prot eomic, or clinical applications. (C) 2001 Academic Press.