A novel experimental design for comparative two-dimensional gel analysis: Two-dimensional difference gel electrophoresis incorporating a pooled internal standard
A novel experimental design for comparative two-dimensional gel analysis: Two-dimensional difference gel electrophoresis incorporating a pooled internal standard
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DOI：
10.1002/pmic.200390006
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发表时间：
2003-01-01
期刊：
影响因子：
3.4
通讯作者：
Currie, I
中科院分区：
文献类型：
--
作者：
Alban, A;David, SO;Currie, I
比较来自不同样品的二维（2-D）凝胶图像是研究蛋白质表达差异的一种既定方法。传统的方法依赖于比较来自至少两种不同凝胶的图像。由于高度变异。二维差异凝胶电泳（etan (TM) DIGE）是一种新兴的比较蛋白质组学技术，可提高样品间差异蛋白表达分析的可重复性和可靠性。在DIGE的应用中，不同的样品用质量和电荷匹配的光谱可分辨荧光染料标记，然后在相同的二维凝胶上分离。我们在大肠杆菌裂解液中加入了不同数量的四种不同的已知蛋白质，通过在每种凝胶中加入一个标准样品，我们测试了一种新的实验设计，该设计利用了DIGE的样品复用能力。标准样品包括等量的待比较样品，并且发现可以提高来自不同凝胶的样品之间蛋白质定量的准确性，从而准确检测样品之间蛋白质水平的微小差异。
The comparison of two-dimensional (2-D) gel images from different samples is an established method used to study differences in protein expression. Conventional methods rely on comparing images from at least 2 different gels. Due to the high variation. Two-dimensional difference gel electrophoresis (Ettan(TM) DIGE) is an emerging technique for comparative proteomics, which improves the reproducibility and reliability of differential protein expression analysis between samples. In the application of DIGE different samples are labelled with mass and charge matched spectrally resolvable fluorescent dyes and are then separated on the same 2-D gel. Using an Escherichia coli lysate "spiked" with varying amounts of four different known proteins, we have tested a novel experimental design that exploits the sample multiplexing capabilities of DIGE, by including a standard sample in each gel. The standard sample comprises equal amounts of each sample to be compared and was found to improve the accuracy of protein quantification between samples from different gels allowing accurate detection of small differences in protein levels between samples.