The practical use of the RFHR 2D PAGE, A new analytical tool for proteomics

O 'Farre II开发的等电点2-D PAGE对酸性至弱碱性蛋白质具有很高的分离能力，已成为蛋白质组学中最常用的方法。但该方法存在识别率低、碱性蛋白分离能力差、人工变性蛋白点多等严重缺陷。这些缺陷阻碍了蛋白质组学的发展。因此，寻找一种新的方法来弥补O 'Farrell法的缺陷是非常重要的，我们研制的RFHR双向电泳法适合于这种需要。该方法对2-D凝胶上检测的蛋白质具有高的基因鉴定率，并且2-D斑点图案具有高的复制能力，适合于自动系统，用于鉴定许多检测的蛋白质的基因，包括不能用O ′ Farrell方法检测的次要蛋白质组分。这种RFHR方法的分离原理与O 'Farrell方法完全不同。的 ...更多信息 RFHR方法的迁移由三个步骤构成，包括0-D迁移，其在1-D迁移之前将蛋白质混合物浓缩到0-D凝胶中的尖锐条带。1-D凝胶没有用于浓缩至等电点的pH梯度，但具有恒定的pH（8.2或9.6），用于通过单个恒定净电荷的恒定迁移速率。二维凝胶没有SDS作为增溶剂，而只有尿素在pH 3.6或3.0下通过天然恒定净电荷迁移，以及18%的凝胶浓度用于分子筛。结果如下：1。我们改进了迁移系统，使其具有更低的温度和更高的电压。扩大了蛋白质点密集分布的区域，可以进行更详细的分析.与Ninon Eido合作开发了商业仪器，并开始主要用于分析碱性蛋白质。3.我们将RFHR方法应用于大肠杆菌的全蛋白质组学研究，已经鉴定出超过324个基因，远远超过了O 'Farrell的方法.我们分析了从稳定期收获的细胞制备的蛋白质，并检测到65个稳定期特异性蛋白质。这些蛋白质的表达时间在稳定期期间彼此不同，这表明一种蛋白质的表达是其他蛋白质表达的触发的可能性。少

The iso-eiectric point 2-D PAGE which O'FarreII developed has a very high separation ability for acidic to weekly basic proteins, and it has been used an the most popular method in proteomics. However, it has several serious defects such as the low identification rate, the low separation ability for basic proteins and many artificial degenerated protein spots. These defects prevent the progress of proteomics. Therefore, finding a new method which makes up for the defects of the O'Farrell's method is important.The RFHR 2-D PAGE which we developed is suitable for this demand. This method has a high gene identification rate for detected proteins on the 2-D gels and the 2-D spot pattern has a high duplication ability appropriate for the automatic system for identifying genes for many detected proteins including minor protein components which can not be detected by the O'Farrell's method. This RFHR method has a separation principle entirely different from that of the O'Farrell's method. The … More migration of the RFHR method is constructed of three steps including the 0-D migration which concentrates protein mixtures to a sharp band into the 0-D gel prior to the 1-D migration. The 1-D gel has no pH gradient for concentration to iso-electric points but a constant pH (8.2 or 9.6) for constant migration rates by individual constant net charges. The 2-D gel baa no SDS as a solubilizer but only urea for migration by native constant net charges at pH 3.6 or 3.0, together with 18 % of gel concentration for molecular sieving.During this Grants-in-Aid, we have further improved the RFHR method, and designed a commercial available apparatus for popular, simplified usage. The results are as follows:1. We improved the migration system to lower temperature and higher voltage. Aa a result the area where a lot of protein spots distribute densely was expanded and can be analyzed in more detail.2. A commercial apparatus was developed in cooperation with Ninon Eido, and began to be used mainly for the analysis of basic proteins..3. We applied the RFHR method to total proteomics in Escherichia coil, and have identified over 324 genes far surpassing the O'Farrell's method.4. We analyzed the proteins prepared from the cells harvested during the stationary phase, and detected 65 stationary phase-specific proteins. The expression times of these proteins are different from each other during the stationary phase suggesting the possibility that the expression of one protein is a trigger for the expression of other proteins. Less