Keck Graduate Institute of Applied Life Sciences, Claremont, CA 91711, USA.
Anal Biochem. 2010 Mar 1;398(1):1-6. doi: 10.1016/j.ab.2009.07.055. Epub 2009 Nov 14.
Fluorescent dyes are widely used for the detection and quantitation of proteins separated by polyacrylamide gel electrophoresis. SYPRO Ruby is one such fluorescent dye widely used for this purpose. More recently, another fluorescent dye, Flamingo, is available for expression proteomic research. Using a standard ultraviolet (UV) transilluminator and a charge-coupled device (CCD)-based imaging system, the relative sensitivity of these two different fluorescent stains with regard to detection of protein spots separated by two-dimensional gel electrophoresis (2D-GE) and identification by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) were compared. Using mouse kidney and liver homogenates as well as Escherichia coli extract, we detected a greater number of protein spots using Flamingo compared with SYPRO Ruby. In addition, when we compared the number of matched peptides and the percentage of amino acid residues identified for 22 different protein spots of mouse kidney proteome, we observed a higher number of matched peptides and a higher percentage of amino acid residues for the majority of the proteins using Flamingo compared with SYPRO Ruby. Also, we were able to characterize a protein spot that can be detected by Flamingo only. Therefore, we recommend Flamingo over SYPRO Ruby to be used for studies on expression proteomics.
荧光染料广泛用于聚丙烯酰胺凝胶电泳分离蛋白质的检测和定量。SYPRO Ruby 就是这样一种广泛用于此目的的荧光染料。最近，另一种荧光染料 Flamingo 可用于表达蛋白质组学研究。使用标准的紫外线（UV）透射仪和基于电荷耦合器件（CCD）的成像系统，比较了这两种不同荧光染料在二维凝胶电泳（2D-GE）分离的蛋白质斑点检测和液相色谱-电喷雾电离-串联质谱（LC-ESI-MS/MS）鉴定方面的相对灵敏度。使用小鼠肾和肝匀浆以及大肠杆菌提取物，我们发现与 SYPRO Ruby 相比，Flamingo 检测到的蛋白质斑点数量更多。此外，当我们比较 22 个不同的小鼠肾蛋白质组蛋白斑点的匹配肽数量和鉴定的氨基酸残基百分比时，我们观察到使用 Flamingo 比 SYPRO Ruby 可以鉴定到更多的匹配肽和更高比例的氨基酸残基。此外，我们还能够对仅用 Flamingo 检测到的蛋白质斑点进行特征分析。因此，我们建议在表达蛋白质组学研究中使用 Flamingo 而不是 SYPRO Ruby。
