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Biotechniques. 2010 Nov;49(5):807-16. doi: 10.2144/000113551.

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

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  • 1Department of Chemistry, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.


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 MutSα on DNA while monitoring MutSα-induced DNA bending using Förster resonance energy transfer (FRET) and to monitor assembly of membrane-tethered SNARE protein complexes.

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