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等电聚焦(IEF)凝胶--多丝支持凝胶(MFS Gel)。MFS凝胶可将聚丙烯酰胺浓度降至2%T(通常为4%T)，从而形成大孔道，从而实现快速等电聚焦，可分离高达200 kDa的高相对分子质量蛋白质。但由于载体两性阳离子的加入形成了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HMW蛋白的pH稳定性和分离度，从而实现对HMW蛋白的快速双向凝胶分离。稳定MFS凝胶的pH梯度。已知固定化pH梯度(IPG)方法即使在较长的IEF时间内也能提供稳定的pH梯度。为了提高凝胶的pH稳定性，将离子交换膜方法引入到MFS凝胶中。制备了IPG-MFS凝胶。为了获得更宽的pH范围和凝胶对凝胶的重现性，还需要对流速控制和仪器改进进行进一步的研究。较低浓度的聚丙烯酰胺凝胶具有较大的孔道，有望提供快速等静电场和HMW蛋白质的分离。热聚合可得到1.75%的T-聚丙烯酰胺，但太易破碎，不适合实际应用。琼脂糖凝胶的加入对低浓度的MFS凝胶有一定的增强作用。辐射是一种促进丙烯酰胺聚合的替代方法，丙烯酰胺能有效地产生自由基。我们利用Co-60伽马射线制备了低于2%T的低浓度MFS凝胶，成功地制备了1.5%T的聚丙烯酰胺，并将其应用于IEF.低浓度MFS凝胶在双向凝胶中的应用低浓度聚丙烯酰胺凝胶在双向凝胶中的应用低浓度的聚丙烯酰胺凝胶在HMW区域提供了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.