Functional characterization and atomic force microscopy of a DNA repair protein conjugated to a quantum dot.
Quantum dots (QDs) possess highly desirable optical properties that make them ideal fluorescent labels for studying the dynamic behavior of proteins. However, a lack of characterization methods for reliably determining protein-quantum dot conjugate stoichiometry and functionality has impeded their widespread use in single-molecule studies. We used atomic force microscopic (AFM) imaging to demonstrate the 1:1 formation of UvrB-QD conjugates based on an antibody-sandwich method. We show that an agarose gel-based electrophoresis mobility shift assay and AFM can be used to evaluate the DNA binding function of UvrB-QD conjugates. Importantly, we demonstrate that quantum dots can serve as a molecular marker to unambiguously identify the presence of a labeled protein in AFM images.
Wang
Hong
H
Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC 27709, USA.
Tessmer
Ingrid
I
Croteau
Deborah L
DL
Erie
Dorothy A
DA
Van Houten
Bennett
B
D001665:Binding Sites; D045643:DNA Repair Enzymes / Q000737:chemistry* / Q000648:ultrastructure*; D018625:Microscopy, Atomic Force / Q000379:methods*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D045663:Quantum Dots
D045643:DNA Repair Enzymes