Proteomic capacity of recent fluorescent dyes for protein staining
Proteomic capacity of recent fluorescent dyes for protein staining
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DOI：
10.1016/j.phytochem.2004.04.019
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发表时间：
2004-06-01
期刊：
影响因子：
3.8
通讯作者：
Rossignol, M
中科院分区：
文献类型：
--
作者：
Chevalier, F;Rofidal, V;Rossignol, M
从分析的蛋白质数量、斑点定量的准确性和重复性来看，二维凝胶染色是比较蛋白质组分析中的关键步骤。在这项工作中，我们比较了最近使用的荧光团对拟南芥总蛋白提取物：SyproRuby(R)(SR)、Deep Purple(R)(DP)和5-十六烷酰氨基荧光素(C16-F)的染色效率。此外，还包括经典的可见染料胶体考马斯蓝(CCB)和硝酸银(SN)。这三种荧光染料获得了高质量的图像，DP提供了更干净的背景，而SR观察到了尖峰，C16-F观察到了粗糙的背景。另一方面，SR和DP的丰富斑点出现饱和。对于相同的蛋白质负载量，在序列SR>；DP中检测到的点数介于CCB的250个和SR的800个之间，与SN>；C16-F>；CCB相近。这些差异主要取决于染料之间的敏感度，从而导致检测到属于较低丰度类别的额外斑点。对复制品斑点的变异系数分布的分析表明，不同染料的染色重复性不同，顺序为SR>；C16-F>；DP>；SN>；CCB。根据具体目标和实际情况，讨论了这些结果对选择方便的着色剂的影响。(C)2004爱思唯尔有限公司。保留所有权利。
Staining of two-dimensional gel constitutes a crucial step in comparative proteome analysis with respect to both the number of proteins analysed, the accuracy of spot quantification and reproducibility. In this work, we compared the efficiency of recent fluorophores to stain Arabidopsis total protein extract: Sypro Ruby(R) (SR), Deep Purple(R) (DP) and 5-hexadecanoylamino-fluorescein (C16-F). In addition, classical visible dyes, colloidal Coomassie blue (CCB) and silver nitrate (SN), were also included. High quality images were obtained for the three fluorescent dyes, DP giving the cleaner background, whereas spikes were observed with SR and a rough background with C16-F. On the other hand, saturation occurred for abundant spots with SR and DP. For a same protein load the number of detected spots ranged between 250 for CCB and 800 for SR in the sequence SR > DP approximate to SN > C16-F > CCB. These differences were shown to rely mainly on the sensitivity between dyes leading to the detection of additional spots belonging to classes of lower abundance. Analysis of the distribution of variation coefficients for spots from replicates showed differences in the staining reproducibility between dyes that ranged in the order SR > C16-F > DP > SN > CCB. The implications of these results for the selection of a convenient stain are discussed according to specific objectives as well as practical aspects. (C) 2004 Elsevier Ltd. All rights reserved.