Development of High-Performance Two-Dimensional Gel Electrophoresis Using Low-Concentration Polyacrylamide Gel Supported by Multifilament Yarns

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We proposed multifilament-supporting gel (MFS gel) as a novel first-dimensional isoelectric focusing (IEF) gel of two-dimensional gel electrophoresis (2-DE). The MFS gel could reduce concentration of polyacrylamide down to 2%T (conventionally 4%T), which would provide large pore of the gel, resulting in fast IEF and separation of high-molecular-weight (HMW) proteins up to 200 kDa. However, due to carrier ampholyte added to form pH gradient, stability and reproducibility was not sufficient. The aim of this research was to improve the pH stability and resolution by optimization of the MFS gel and reduction of polyacrylamide gel concentration, to obtain fast 2-DE separation of HMW proteins.1. Stabilize pH gradient of the MFS gel.Immobilized pH gradient (IPG) method is known that it provides a stable pH gradient during even a long IEF time. To improve pH stability, IEF method was introduced to the MFS gel. IPG MFS gel was prepared. Further investigation on controlling flow rate and instrumental improvement was still necessary to obtain wider pH range and gel-to-gel reproducibility.2. Lower the MFS gel concentrationPolyacrylamide gel of lower concentration has larger pore and is expected to provide fast IEF and separation of HMW proteins. Polymerization by heat could provide 1.75%T polyacrylamide, but too fragile for practical use. Addition of agarose was found to be effective to reinforce the low-concentration MFS gel to some degree. Radiation is an alternative to promote polymerization of acrylamide, which effectively produces radicals. We utilized Co-60 gamma ray to prepare MFS gels of low concentration below 2%T. Polyacrylamide of 1.5%T could be successfully prepared and applied to IEF.3. Application of low-concentration MFS gel to 2-DEDeveloped low-concentration polyacrylamide gel was applied to 2-DE. The low-concentration polyacrylamide gels were proved to provide clearer protein spots in the HMW region.