One protein/cell proteomics by the high power RFHR-2D-PAGE

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The IPG method based on O'Farrell's iso-electric point electrophoresis is exclusively popular on proteomics. The IPG method, however, has two serious weak points, and has brought about a difficulty to the protein separation step. These weak points are that the IPG method can not separate basic proteins sufficiently, and that this method makes proteins split on 2D gels to about ten spots. To conquer these weak points, a new method is necessary, which has a new separation principle different from that of the IPG method. The RFHR 2D PAGE can satisfy the request, because it does not adopt the iso-electric point electrophoresis. The aim of this project is to improve the separation ability for detection and identification of extremely small amount proteins, and to carry out proteomics for E.coli and eukaryotes by the improved RFHR method. First, on the increase of separation ability of the RFHR method We developed a water- cooled apparatus to control temperature strictly. The separation ability increased greatly, and we identified 568 genes for E.coli proteins larger than two times as much as those by the IPG method. This identified number corresponds to 80% of 700 protein spots detected with CBB staining. The 0D electrophoresis for protein concentration was abolished, and the proteins were concentrated into the top of the 1D gels using the 0D concentration buffers, prior to the 1D electrophoresis. By this trial, future improvement of the RFHR method to automatic and convenient operations will become more easy. Secondly, on proteomics of eukaryotesA difficult point on rat liver proteomics is to increase solubility of proteins. We introduced 2 M thio-urea to the 0D and 1D gels. This treatment solubilized larger and membrane bound proteins more effectively. We began human proteomics from the 3^<rd> year, and will continue it from now on.