Quantum dots (QDs) are extremely bright fluorescent imaging probes that are particularly useful for tracking individual molecules in living cells. Here, we show how a two-component system composed of a high-affinity single-chain fragment antibody and its cognate hapten (fluorescein) can be utilized for tracking individual proteins in various cell types. The single-chain fragment antibody against fluorescein is genetically appended to the protein of interest, while the hapten fluorescein is attached to the end of the peptide that is used to coat the QDs. We describe (i) the method used to functionalize QDs with fluorescein peptides; (ii) the method used to control the stoichiometry of the hapten on the surface of the QD; and (iii) the technical details necessary to observe single molecules in living cells.
|Title of host publication||Methods in Enzymology|
|Publisher||Academic Press Inc.|
|Number of pages||19|
|State||Published - 2010|
|Name||Methods in Enzymology|
Bibliographical noteFunding Information:
We thank Professor Wittrup for providing the 4M5.3 scFv plasmid, Professor Lindquist for the mouse PrP plasmid, and Dr. Thomas Dertinger for help with FACS data import. We acknowledge the help of Dr. Ingrid Schmid, supervisor of the UCLA Jonsson Comprehensive Cancer Center (JCCC) and Center for AIDS Research Flow Cytometry Core Facility that is supported by National Institutes of Health awards CA-16042 and AI-28697, and by the JCCC, the UCLA AIDS Institute, and the David Geffen School of Medicine at UCLA. Ensemble confocal imaging was performed at the UCLA/CNSI Advanced Light Microscopy Shared Facility. This work was supported by NIH/NIBIB BRP Grant 5-R01-EB000312 and the NSF, The Center for Biophotonics, an NSF Science and Technology Center managed by the University of California, Davis, under Cooperative Agreement PHY0120999.