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

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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