I am a little hesitant to respond to Michael Kuhn statement that there is "little reason to switch to these much more expensive, proprietary dyes". He has only listed one advantage of Alexa Fluor 488 and Oregon Green 488 have over fluorescein and 5,(6)-carboxyfluorescein. I do not want to turn this into marketing pitch but Alexa Fluor 488 and Oregon Green 488 has several other distinct and key attributes. All of this information is freely available on our web site including additional references and figures. Fluorescein dyes, including 5,(6)-carboxyfluorescein, and their conjugates have several drawbacks, including 1. A relatively high rate of photobleaching 2 The pka of fluorescein is 6.4 . This means that the fluorescence of fluorescein is significantly reduced below pH 7. 3. A tendency toward quenching on conjugation to biopolymers, particularly at high degrees of substitution The photobleaching and pH sensitivity of fluorescein make quantitative measurements with this fluorophore problematic. Alexa Fluor 488 1. pH-insensitive fluorescence between pH 4 and 10. 2. Almost spectrally identical to fluorescein, with excitation/emission maxima of 491/515 nm. 3. Strong absorption, with an extinction coefficient greater than 65,000 cm-1M-1. 4. Much more photostable than fluorescein, allowing more time for observation and image capture. 5. Alexa Fluor 488 succinimidyl ester is water soluble, requiring no organic co-solvents in labeling reactions, suggesting that the Alexa Fluor 488 succinimidyl ester may be the ideal reagent for labeling exposed cell-surface proteins of live cells. 6. Superior fluorescence output per protein conjugate, surpassing that of any other spectrally similar fluorophore-labeled protein, including fluorescein conjugates. Oregon Green Dyes Bioconjugates prepared from the Oregon Green 488 and Oregon Green 514 dyes share several advantages over those of other fluorescein dyes. These include: 1. Fluorescence of protein conjugates prepared from the Oregon Green 488 and Oregon Green 514 dyes is not appreciably quenched, even at high degrees of labeling. 2. Conjugates of the Oregon Green 488 and Oregon Green 514 fluorophores are more photostable than those of fluorescein or Oregon Green 488 dyes). The superior photostability of Oregon Green 488 dye and, in particular, the Oregon Green 514 conjugates permit the acquisition of many more photons before the photodestruction of the dye, making the Oregon Green dyes particularly useful substitutes for fluoresceins for fluorescence imaging applications. 3. The Oregon Green dyes have a lower pKa (pKa = 4.7 versus 6.4 for fluorescein), making their fluorescence essentially pH insensitive in the physiological pH range. 4. The pH sensitivity of the Oregon Green dyes in the weakly acidic range (pH 4-6) also makes these dyes useful as pH indicators for acidic organelles of live cells Helix Research wrote: > Wolfgang, The only advantages of Oregon Green 488 and Alexa 488 are > that they are more photostable than FITC. If photostability is not an > issue and you are able to get good results with FITC, there is little > reason to switch to these much more expensive, proprietary dyes. One > altrnative to FITC is 5,(6)-carboxyfluorescein, NHS ester. This is an > amine-reactive fluorescein that results in a more stable (amide) > linkage than the thiourea bond formed when labeling amines with > fluorescein isothiocyanate. Best regards, Michael Kuhn, PresidentHelix > ResearchFluorescence > Chemistrymkuhn@helixresearch.comwww.helixresearch.comPhone: 541 988 > 0464FAX: 541 744 9179 -- Robert Archer, Ph.D. Cell Biology Product Manager mailto:robert@probes.com Molecular Probes, Inc. 4849 Pitchford Ave Eugene, OR 97402-9165 Tel: (541) 465-8353 Fax: (541) 465-4593 http://www.probes.com
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