title: Quantitative comparison of long-wavelength Alexa Fluor dyes to Cy dyes: fluorescence of the dyes and their bioconjugates.
abstract: Amine-reactive N-hydroxysuccinimidyl esters of Alexa Fluor fluorescent dyes with principal absorption maxima at about 555 nm, 633 nm, 647 nm, 660 nm, 680 nm, 700 nm, and 750 nm were conjugated to antibodies and other selected proteins. These conjugates were compared with spectrally similar protein conjugates of the Cy3, Cy5, Cy5.5, Cy7, DY-630, DY-635, DY-680, and Atto 565 dyes. As N-hydroxysuccinimidyl ester dyes, the Alexa Fluor 555 dye was similar to the Cy3 dye, and the Alexa Fluor 647 dye was similar to the Cy5 dye with respect to absorption maxima, emission maxima, Stokes shifts, and extinction coefficients. However, both Alexa Fluor dyes were significantly more resistant to photobleaching than were their Cy dye counterparts. Absorption spectra of protein conjugates prepared from these dyes showed prominent blue-shifted shoulder peaks for conjugates of the Cy dyes but only minor shoulder peaks for conjugates of the Alexa Fluor dyes. The anomalous peaks, previously observed for protein conjugates of the Cy5 dye, are presumably due to the formation of dye aggregates. Absorption of light by the dye aggregates does not result in fluorescence, thereby diminishing the fluorescence of the conjugates. The Alexa Fluor 555 and the Alexa Fluor 647 dyes in protein conjugates exhibited significantly less of this self-quenching, and therefore the protein conjugates of Alexa Fluor dyes were significantly more fluorescent than those of the Cy dyes, especially at high degrees of labeling. The results from our flow cytometry, immunocytochemistry, and immunohistochemistry experiments demonstrate that protein-conjugated, long-wavelength Alexa Fluor dyes have advantages compared to the Cy dyes and other long-wavelength dyes in typical fluorescence-based cell labeling applications.
authors: Molecular Probes, Inc., Eugene, Oregon 97402, USA.
authors: Judith E
authors: JE
authors: Berlier
authors: Anca
authors: A
authors: Rothe
authors: Gayle
authors: G
authors: Buller
authors: Jolene
authors: J
authors: Bradford
authors: Diane R
authors: DR
authors: Gray
authors: Brian J
authors: BJ
authors: Filanoski
authors: William G
authors: WG
authors: Telford
authors: Stephen
authors: S
authors: Yue
authors: Jixiang
authors: J
authors: Liu
authors: Ching-Ying
authors: CY
authors: Cheung
authors: Wesley
authors: W
authors: Chang
authors: James D
authors: JD
authors: Hirsch
authors: Joseph M
authors: JM
authors: Beechem
authors: Rosaria P
authors: RP
authors: Haugland
authors: Richard P
authors: RP
authors: Haugland
mesh_terms: D000818:Animals; D001578:Benzopyrans / Q000737:chemistry; D001921:Brain / Q000648:ultrastructure; D002232:Carbocyanines / Q000737:chemistry; D002417:Cattle; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000166:cytology / Q000648:ultrastructure; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D007150:Immunohistochemistry; D007211:Indoles / Q000737:chemistry; D008856:Microscopy, Fluorescence; D025261:Mitochondrial Proton-Translocating ATPases / Q000378:metabolism; D038761:Photobleaching; D011506:Proteins / Q000737:chemistry*; D011651:Pulmonary Artery / Q000166:cytology / Q000201:enzymology / Q000648:ultrastructure; D051381:Rats; D012995:Solubility; D013053:Spectrophotometry; D013386:Succinates / Q000737:chemistry; D013601:T-Lymphocytes / Q000166:cytology; D014867:Water
chemical_list: D001578:Benzopyrans; D002232:Carbocyanines; D005456:Fluorescent Dyes; D007211:Indoles; D011506:Proteins; D013386:Succinates; C021983:complex V (mitochondrial oxidative phosphorylation system); D014867:Water; D025261:Mitochondrial Proton-Translocating ATPases