Fluorescent DNA hybridization probe preparation using amine modification and reactive dye coupling.

Fluorescent nucleic acid hybridization probes traditionally have been generated by enzymatic incorporation of dye-labeled nucleotides, even though incorporation efficiency is low and variable from dye to dye. Alternatively, 5-(3-aminoallyl)-2'-deoxyuridine 5'-triphosphate (aa-dUTP) is enzymatically incorporated to generate amine-modified DNA, which is then chemically labeled with an amine-reactive fluorescent dye. We optimized this latter two-step approach for maximal hybridization signal brightness using DNA probes labeled to varying degrees with different fluorescent dyes.

Development and characterization of the NanoOrange protein quantitation assay: a fluorescence-based assay of proteins in solution.

We developed a sensitive fluorescence assay for the quantitation of proteins in solution using the NanoOrange reagent, a merocyanine dye that produces a large increase in fluorescence quantum yield upon interaction with detergent-coated proteins. The NanoOrange assay allowed for the detection of 10 ng/mL to 10 micrograms/mL protein with a standard fluorometer, offering a broad, dynamic quantitation range and improved sensitivity relative to absorption-based protein solution assays. The protein-to-protein variability of the NanoOrange assay was comparable to those of standard assays, including Lowry, bicinchoninic acid, and Bradford procedures.