Berlier Judith E, Rothe Anca, Buller Gayle, Bradford Jolene, Gray Diane R, Filanoski Brian J, Telford William G, Yue Stephen, Liu Jixiang, Cheung Ching-Ying, Chang Wesley, Hirsch James D, Beechem Joseph M, Haugland Rosaria P, Haugland Richard P
Molecular Probes, Inc., Eugene, Oregon 97402, USA.
J Histochem Cytochem. 2003 Dec;51(12):1699-712. doi: 10.1177/002215540305101214.
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.
具有约555nm、633nm、647nm、660nm、680nm、700nm和750nm主要吸收峰的Alexa Fluor荧光染料的胺反应性N - 羟基琥珀酰亚胺酯与抗体及其他选定蛋白质进行了共轭。将这些共轭物与Cy3、Cy5、Cy5.5、Cy7、DY - 630、DY - 635、DY - 680和Atto 565染料的光谱相似的蛋白质共轭物进行了比较。作为N - 羟基琥珀酰亚胺酯染料，Alexa Fluor 555染料在吸收峰、发射峰、斯托克斯位移和消光系数方面与Cy3染料相似，Alexa Fluor 647染料与Cy5染料相似。然而，这两种Alexa Fluor染料比其相应的Cy染料对光漂白具有显著更高的抗性。由这些染料制备的蛋白质共轭物的吸收光谱显示，Cy染料共轭物有明显的蓝移肩峰，而Alexa Fluor染料共轭物只有较小的肩峰。先前在Cy5染料的蛋白质共轭物中观察到的异常峰可能是由于染料聚集体的形成。染料聚集体对光的吸收不会产生荧光，从而降低了共轭物的荧光。蛋白质共轭物中的Alexa Fluor 555和Alexa Fluor 647染料表现出明显更少的这种自猝灭现象，因此Alexa Fluor染料的蛋白质共轭物比Cy染料的蛋白质共轭物具有显著更强的荧光，尤其是在高标记度时。我们的流式细胞术、免疫细胞化学和免疫组织化学实验结果表明，在典型的基于荧光的细胞标记应用中，蛋白质共轭的长波长Alexa Fluor染料比Cy染料和其他长波长染料具有优势。
