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首页> 外文期刊>Cytometry: The Journal of the Society for Analytical Cytology >Phycoerythrin-allophycocyanin: A resonance energy transfer fluorochrome for immunofluorescence
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Phycoerythrin-allophycocyanin: A resonance energy transfer fluorochrome for immunofluorescence

机译：藻红蛋白-铝藻蓝蛋白：用于免疫荧光的共振能量转移荧光染料

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Background: As immunofluorescence experiments become more complex, the demand for new dyes with different properties increases. Fluorescent dyes fifth large Stoke's shifts that are very bright and have low background binding to cells are especially desirable. We report on the properties of the resonance energy tandems of phycoerythrin and allophycocyanin (PE-APC). PE-APC is the original fluorescence resonance energy tandem dye described in the literature, but it has not been utilized because of the difficulty of synthesizing and preparing a consistent product. Methods: PE-APC complexes comprising different ratios of the two phycobiliproteins conjugated to streptavidin were synthesized using standard protein-protein conjugation chemistry. The PE-APC streptavidins were evaluated for flow cytometric analysis. They were compared directly to Cy5PE conjugates because Cy5PE is the fluorophore that is spectrally most like the PE-APC. Results: PE-APC complexes showed the expected fluorescence spectral properties of a tandem: excitation was excellent at 488 nm (and best at the PE excitation maximum) and emission was greatest at the APC emission maximum at about 660 nm. The efficiency of transfer of energy from PE to APC was about 90%. Conclusion: PE-APC can be considered an excellent substitute for Cy5PE. Compared with Cy5PE, PE-APC has similar brightness (in staining experiments), slightly greater compensation requirements with PE but much lower compensation with Cy5.5PE or Cy5.5PerCP, and lower nonspecific background binding. PE-APC is a useful alternative to Cy5PE, especially in applications in which the use of Cy5 is impractical. Cytometry 44:24 -29, 2001. Published 2001 Wiley-Liss, Inc.(dagger). [References: 3]
机译：背景：随着免疫荧光实验变得越来越复杂，对具有不同特性的新型染料的需求也在增加。非常理想的是荧光染料的第五大斯托克位移，其非常明亮并且与细胞的背景结合力低。我们报告了藻红蛋白和别藻蓝蛋白（PE-APC）的共振能量tandems的特性。 PE-APC是文献中描述的原始荧光共振能量串联染料，但由于难以合成和制备一致的产物而未被使用。方法：采用标准的蛋白质-蛋白质缀合化学方法合成包含不同比例的两种链亲和素的两种藻胆蛋白的PE-APC复合物。评价PE-APC链霉亲和素用于流式细胞仪分析。将它们直接与Cy5PE缀合物进行比较，因为Cy5PE是光谱上最像PE-APC的荧光团。结果：PE-APC配合物显示了预期的串联荧光光谱特性：在488 nm处激发极佳（在PE激发最大值处最佳），在APC发射最大值处约660 nm处激发最大。从PE到APC的能量转移效率约为90％。结论：PE-APC被认为是Cy5PE的优良替代品。与Cy5PE相比，PE-APC具有相似的亮度（在染色实验中），PE的补偿要求略高，但Cy5.5PE或Cy5.5PerCP的补偿低得多，并且非特异性背景结合也更低。 PE-APC是Cy5PE的有用替代品，特别是在其中不使用Cy5的应用中。 Cytometry 44：24 -29，2001。2001 Wiley-Liss，Inc。（匕首）出版。 [参考：3]

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