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

提出了一种新型的二维凝胶电泳（2-DE）的一维等电聚焦（IEF）凝胶。MFS凝胶可以将聚丙烯酰胺的浓度降低到2%T（传统的4%T），这将提供大的凝胶孔，从而实现快速IEF和高达200 kDa的高分子量（HMW）蛋白质的分离。但由于载体两性电解质的加入形成pH梯度，稳定性和重现性不够。本研究的目的是通过优化MFS凝胶和降低聚丙烯酰胺凝胶浓度来提高pH稳定性和分辨率，以实现HMW蛋白的快速2-DE分离。稳定MFS凝胶pH梯度。固定化pH梯度（IPG）法具有在较长时间内保持稳定的pH梯度的优点。为了提高pH稳定性，将IEF法引入到MFS凝胶中。制备了IPG MFS凝胶。为了获得更宽的pH范围和凝胶间的重现性，还需要进一步研究控制流速和改进仪器。降低MFS凝胶浓度低浓度的聚丙烯酰胺凝胶具有更大的孔隙，有望提供快速的IEF和HMW蛋白分离。热聚合可以提供1.75%T的聚丙烯酰胺，但太脆弱，无法实际使用。琼脂糖的加入对低浓度MFS凝胶有一定的强化作用。辐射是促进丙烯酰胺聚合的一种替代方法，可以有效地产生自由基。我们利用Co-60伽马射线制备了低于2%T的低浓度MFS凝胶。成功制备了1.5%T的聚丙烯酰胺，并应用于ie3。低浓度MFS凝胶在2-DE中的应用研制了低浓度聚丙烯酰胺凝胶在2-DE中的应用。低浓度聚丙烯酰胺凝胶被证明在HMW区域提供更清晰的蛋白质斑点。

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.