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Detection of Epitope-Tagged Proteins in Flow Cytometry: Fluorescence Resonance Energy Transfer-Based Assays on Beads with Femtomole Resolution by Tione Buranda; Gabriel P. Lopez; Peter Simons; Andrzej Pastuszyn; Larry A. Sklar is a Biochemistry, Genetics and Molecular Biology article available to read on EtoBox.

What is Detection of Epitope-Tagged Proteins in Flow Cytometry: Fluorescence Resonance Energy Transfer-Based Assays on Beads with Femtomole Resolution about?

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 K d 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 K d values in the 0.3-2.0 nM range. The affini

Who reads Detection of Epitope-Tagged Proteins in Flow Cytometry: Fluorescence Resonance Energy Transfer-Based Assays on Beads with Femtomole Resolution?

Author

Tione Buranda; Gabriel P. Lopez; Peter Simons; Andrzej Pastuszyn; Larry A. Sklar

Publisher

Elsevier Science; Elsevier ; Elsevier Inc.; Elsevier BV (ISSN 0003-2697)

Published

2001

Language

EN

Field

Biochemistry, Genetics and Molecular Biology (Life Sciences)