* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download DNA and Protein Concentration Measurements Using Fluorescence
Protein moonlighting wikipedia , lookup
Gel electrophoresis of nucleic acids wikipedia , lookup
Non-coding DNA wikipedia , lookup
Comparative genomic hybridization wikipedia , lookup
Protein (nutrient) wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Deoxyribozyme wikipedia , lookup
History of molecular evolution wikipedia , lookup
Molecular cloning wikipedia , lookup
Surround optical-fiber immunoassay wikipedia , lookup
Community fingerprinting wikipedia , lookup
Protein adsorption wikipedia , lookup
DNA vaccination wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Real-time polymerase chain reaction wikipedia , lookup
Protein–protein interaction wikipedia , lookup
Circular dichroism wikipedia , lookup
Molecular Inversion Probe wikipedia , lookup
Nuclear magnetic resonance spectroscopy of proteins wikipedia , lookup
Molecular evolution wikipedia , lookup
Western blot wikipedia , lookup
THE APPLICATION NOTEBOOK – SEPTEMBER 2006 ADVERTISING SUPPLEMENT Molecular Spectroscopy | 21 DNA and Protein Concentration Measurements Using Fluorescence Analysis Yvette Mattley and Jay Thomason, Ocean Optics The Ocean Optics Curie Spectrofluorometer is designed to measure fluorophores with excitation and emission properties across a wide 200-850 nm UV-Vis wavelength range. Our objective is to demonstrate the capabilities and sensitivity of the system in measuring DNA and protein concentration in solution using three Molecular Probes Quant-iT Assay Kits. Detection limits are reported for each kit. T he Ocean Optics Curie is a high-sensitivity cuvette emission spectrofluorometer that can detect picomolar-range concentration of fluorophores in solution from 200–850 nm. The Curie features a CCD-array detector and is distinguished by internal linear variable filtering technology. The patented filtering helps to discriminate between powerful pulsed xenon wavelengths from the onboard excitation source and the weak spectral emissions from samples. The Curie is the only spectrofluorometer with linear variable filtering technology that can be used to spectrally shape excitation energy and eliminate the need for scanning monochromators. Each filter’s transmission or blocking band can be moved throughout the 230–500 nm or 300–750 nm wavelength range. Experimental Conditions Three Molecular Probes Quant-iT Assay Kits were used to review the Curie’s performance. The Quant-iT PicoGreen dsDNA Assay Kit (P7589) and the DNA Broad Range DNA Assay Kit (Q33130) were used to measure double-stranded DNA (dsDNA) in solution. The Quant-iT Protein Assay Kit (Q33210) was used to measure protein in solution. All working solutions and standards were prepared per the instructions provided with each kit. Sufficient volumes were prepared to allow for measurements in a standard 1-cm quartz fluorescence cuvette. to 10 ng/mL concentration at a five-second integration time. Even lower detection limits are possible with longer integration periods. Figure 2 Figure 2: Fluorescence spectra acquired for the shows the fluo- Broad Range DNA Assay Kit (Molecular Probes Quant-iT Broad Range dsDNA Kit [Q33130]). rescence spectra acquired for the Broad Range DNA Assay Kit. Using this kit, the Curie was able to detect dsDNA to 5 mg/mL concentration at a 1-s integration time. Figure 3 shows the fluorescence spectra acquired for the Protein Assay Kit. Using this kit, the Curie was able to detect protein concentration to 25 mg/mL at a 0.5-s integration time. Conclusions The Curie Spectrofluorometer is sensitive enough to detect the full range of fluorescence standards included in the Quant-iT DNA and Protein Assay Kits. Using the ultra-sensitive PicoGreen Assay Kit, detection limits to 10 ng/mL were achieved. These data illustrate that the Curie is capable of excitation and emission detection throughout the UV-Vis region. The Curie is a cost-effective CCD-array platform that provides a less complex alternative to more sensitive PMT-based technologies for dsDNA and protein analysis. Results Figure 1 shows the fluorescence spectrum acquired for the Pico-Green Assay Kit. Using this ultra-sensitive kit, the Curie Figure 1: Fluorescence spectra acquired for the was able to PicoGreen Assay Kit (Molecular Probes Quantdetect dsDNA iT PicoGreen dsDNA Kit [P7589]). Figure 3: Fluorescence spectra acquired for the Protein Assay Kit (Molecular Probes Quant-iT Protein Assay Kit [Q33210]). Ocean Optics 830 Douglas Avenue, Dunedin, FL 34698 USA Tel. (727) 733-2447, Fax (727) 733-3962 [email protected], www.oceanoptics.com For More Information Circle 13