Mahon P, Dupree P
University of Cambridge, Department of Biochemistry, United Kingdom.
Electrophoresis. 2001 Jun;22(10):2075-85. doi: 10.1002/1522-2683(200106)22:10<2075::AID-ELPS2075>3.0.CO;2-C.
Quantitative two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is used to determine changes in individual protein levels in complex protein mixtures. To provide reliable data, the software used for 2-D gel image analysis must provide a linear response over a wide dynamic range of data output. Here, we show that Phoretix 2D Full analysis of 2-D gels stained with colloidal Coomassie Brilliant Blue G-250 can provide a linear measure of changes in protein quantity. We show using a complex mixture of Arabidopsis thaliana proteins, that this is true for essentially all focused proteins, in a data output range greater than three orders of magnitude. An analysis of the factors that affect errors in the results demonstrated that reproducibility of the data is significantly improved by user seeding, whereas it is reduced by use of the background subtraction algorithms.
定量二维聚丙烯酰胺凝胶电泳（2-D PAGE）用于确定复杂蛋白质混合物中单个蛋白质水平的变化。为了提供可靠的数据，用于二维凝胶图像分析的软件必须在宽动态范围的数据输出上提供线性响应。在此，我们表明，用考马斯亮蓝G-250胶体染色的二维凝胶的Phoretix 2D全分析可以提供蛋白质数量变化的线性测量。我们使用拟南芥蛋白质的复杂混合物表明，在大于三个数量级的数据输出范围内，基本上所有聚焦的蛋白质都是如此。对影响结果误差的因素的分析表明，用户接种可显著提高数据的重现性，而使用背景扣除算法则会降低数据的重现性。
