HiLyte Fluor - Eurogentec

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6 HiLyte Fluor 647 Dye, an Excellent Replacement for Cy5 Dye Cy5 is the preferred dye for preparing near-infrared fluorescent bioconjugates. Compared to the spectra of Cy5 dyes, the spectra of HiLyte Fluor 647 conjugates are only slightly red-shifted. This slight change in absorption spectrum makes HiLyte Fluor 647 dyes an optimal match of filters designed for Cy5 dyes. In side-by-side comparison of antibody conjugates of HiLyte Fluor 647 dyes and Cy5 conjugates (supplied by other companies), the total fluorescence of HiLyte Fluor 647 labeled secondary antibodies is is significantly higher than that of Cy5 conjugates. Extinction coefficient is 250,000 M -1 cm -1 , with a quantum yield of 0.33. Unlike Cy5 dyes, HiLyte Fluor 647 dyes have very little change in absorption or fluorescence spectra when conjugated to most proteins, oligonucleotides and nucleic acids, thus yielding greater total fluorescence at the same degree of substitution. Normalized Absorbance 1.2 1.0 0.8 0.6 0.4 0.2 A 0.0 500 600 700 0.0 800 Wavelength (nm) Product Size Cat# HiLyte Fluor 647 acid 5 mg 81255 HiLyte Fluor 647 amine 1 mg 81257 HiLyte Fluor 647 C2 maleimide 1 mg 81259 HiLyte Fluor 647 hydrazide 1 mg 81258 HiLyte Fluor 647 succinimidyl ester (SE) 1 mg 81256 1.2 1.0 0.8 0.6 0.4 0.2 Normalized Fluorescence RFU 35000 25000 15000 5000 HiLyte Fluor 647 Alexa Fluor 647 DyLight 647 Cy5 0 0 200 400 600 Rabbit IgG (ng/well) DC DE Figure 7. Absorption and emission spectra of HiLyte Fluor 647 (panel A). Performance of dye-goat anti-rabbit IgG conjugates (panel B). HiLyte Fluor 647 and Alexa Fluor 647 photostability over time (panel C). 3T3 cells incubated with anti-tubulin antibody and goatanti-rabbit antibodies, labeled either with HiLyte Fluor 647 (panel D), or Cy5 (panel E), nuclei were stained with Hoechst 33342 (Cat# 83218). Figure 8. α-Tubulins of bovine pulmonary artery endothelial cells were probed with biotin-conjugated mouse anti-α-tubulin, visualized with HiLyte Fluor 647 conjugated streptavidin (Cat# 60667). Mitochondria were detected with mouse anti-Oxphos V complex, visualized with HiLyte Fluor 488-conjugated goat anti-mouse (Cat# 28175-H488), nuclei stained with DAPI (Cat# 83210). 0.4 0 50 100 150 200 250 300 Irradiation Time (min) © 2009 AnaSpec, Inc. • 2149 O’Toole Ave. • San Jose, CA 95131 • 800-452-5530 • www.anaspec.com B A/A 0 1.2 1.0 0.8 0.6 C HiLyte Fluor TM 647 Alexa Fluor TM 647
HiLyte Fluor 680 Dye, an Excellent Replacement for Cy5.5 Dye With a peak excitation at 678 nm and emission maximum at 699 nm, HiLyte Fluor 680 dyes are spectrally similar to Cy5.5 dyes. Fluorescence emission of HiLyte Fluor 680 dyes is well separated from that of other commonly used red fluorophores, such as TAMRA, R-phycoerythrin and HiLyte Fluor 647 dyes, making it ideal for three and four-color labeling. Extinction coefficient is 190,000 M -1 cm -1 , with a quantum yield of 0.36. Product Size Cat# HiLyte Fluor 680 acid 5 mg 81260 HiLyte Fluor 680 amine 1 mg 81262 HiLyte Fluor 680 C2 maleimide 1 mg 81264 HiLyte Fluor 680 hydrazide 1 mg 81263 HiLyte Fluor 680 succinimidyl ester (SE) 1 mg 81261 Normalized Absorbance 1.2 1.0 0.8 0.6 0.4 0.2 0.0 600 700 Wavelength (nm) 800 © 2009 AnaSpec, Inc. • 2149 O’Toole Ave. • San Jose, CA 95131 • 800-452-5530 • www.anaspec.com 1.2 1.0 0.8 0.6 0.4 0.2 0.0 Normalized Flurescence Figure 9. Absorption and emission spectra of HiLyte Fluor 680 HiLyte Fluor Fluor Dyes Spanning the spectrum Figure 10. α-Tubulin in 3T3 cells probed with mouse anti-tubulin, visualized with HiLyte Fluor 680-conjugated rabbit-anti-mouse IgG (Cat# 28164-H680), nuclei stained with DAPI (Cat# 83210). 7
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