原创力文档Tracking Single Proteins in Live Cells Using Single Chain Antibody Fragment Fluorescent Quantum Dot Affinity Pair英文文献.pdfVIP
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C H A P T E R T H R E E
Tracking Single Proteins in Live
Cells Using Single-Chain Antibody
Fragment-Fluorescent Quantum
Dot Affinity Pair
Gopal Iyer, Xavier Michalet, Yun-Pei Chang, and Shimon Weiss
Contents
1. Introduction 62
2. The Method: Targeting QDs via a Single-Chain Variable
Fragment-Hapten Pair 64
3. Functionalization of QDs 66
3.1. Reagents for peptide coating 66
3.2. Peptide coating of QDs 66
3.3. Varying the stoichiometry of FL-pc-QDs 68
4. Quantification of the Number of FL Molecules per FL-pc-QD 69
5. Binding of FL-QDs to Anti-scFv Fusion Constructs 71
5.1. Quantification of the binding affinity of FL-QD to a-FL scFv 71
6. DNA Constructs for Single FL-QD Imaging in Live Cells 73
6.1. Labeling of live mammalian cells 74
7. Single-Molecule Imaging of Live Mammalian Cells 74
Acknowledgments 75
References 77
Abstract
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
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