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Quantitative comparison of long-wavelength Alexa Fluor dyes to Cy dyes: fluorescence of the dyes and their bioconjugates.

The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society (2003-11-19)
Judith E Berlier, Anca Rothe, Gayle Buller, Jolene Bradford, Diane R Gray, Brian J Filanoski, William G Telford, Stephen Yue, Jixiang Liu, Ching-Ying Cheung, Wesley Chang, James D Hirsch, Joseph M Beechem, Rosaria P Haugland, Richard P Haugland
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.

MATERIALS
Product Number
Brand
Product Description

Supelco
Atto 565 NHS ester, BioReagent, suitable for fluorescence, ≥70.0% (coupling to amines)
Sigma-Aldrich
Atto 647 NHS ester, BioReagent, suitable for fluorescence, ≥80% (coupling to amines)
Sigma-Aldrich
Atto 633, BioReagent, suitable for fluorescence
Sigma-Aldrich
Atto 565 maleimide, BioReagent, suitable for fluorescence
Sigma-Aldrich
Atto 565-Streptavidin, BioReagent, suitable for fluorescence
Sigma-Aldrich
Dy-677 NHS ester, for fluorescence
SAFC
Dy-554 NHS ester, for fluorescence
Sigma-Aldrich
Dy-549 NHS ester, for fluorescence
Sigma-Aldrich
Atto 633 Protein Labeling Kit
Sigma-Aldrich
Atto 700 maleimide, BioReagent, suitable for fluorescence, ≥90% (coupling to thiols)
Sigma-Aldrich
Atto 700, BioReagent, suitable for fluorescence, ≥96.0% (HPCE)

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