Four-color single-molecule fluorescence with noncovalent dye labeling to monitor dynamic multimolecular complexes

Autor(en): DeRocco, Vanessa C.
Anderson, Trevor
Piehler, Jacob 
Erie, Dorothy A.
Weninger, Keith
Stichwörter: ALTERNATING-LASER EXCITATION; Biochemical Research Methods; Biochemistry & Molecular Biology; DNA mismatch repair; FRET; FUSION; HISTIDINE-TAGGED PROTEINS; MECHANISM; Msh2; Msh6; Munc-18; MutS; REAL-TIME; RECOGNITION; RESONANCE ENERGY-TRANSFER; SNARE COMPLEX; SNARE proteins; SPECTROSCOPY
Erscheinungsdatum: 2010
Herausgeber: FUTURE SCI LTD
Journal: BIOTECHNIQUES
Volumen: 49
Ausgabe: 5
Startseite: 807+
Zusammenfassung: 
To enable studies of conformational changes within multimolecular complexes, we present a simultaneous, four-color single molecule fluorescence methodology implemented with total internal reflection illumination and camera-based, wide-field detection. We further demonstrate labeling histidine-tagged proteins noncovalently with Tris-nitrilotriacetic acid (Tris-NTA)-conjugated dyes to achieve single molecule detection. We combine these methods to colocalize the mismatch repair protein MutS alpha. on DNA while monitoring MutS alpha-induced DNA bending using Forster resonance energy transfer (FRET) and to monitor assembly of membrane-tethered SNARE protein complexes.
ISSN: 07366205
DOI: 10.2144/000113551

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