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

本文提出了一种新型的二维凝胶电泳一维等电聚焦（IEF）凝胶--多功能支持凝胶（MFS凝胶）。MFS凝胶可以将聚丙烯酰胺浓度降低至2%T（常规为4%T），这将提供大孔的凝胶，从而导致快速IEF和高达200 kDa的高分子量（HMW）蛋白的分离。然而，由于添加载体两性电解质以形成pH梯度，稳定性和再现性不足。本研究的目的是通过优化MFS凝胶的pH稳定性和降低聚丙烯酰胺凝胶的浓度来提高其分辨率，实现HMW蛋白质的快速双向电泳分离.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。为了改善pH稳定性，将IEF方法引入MFS凝胶。制备IPG MFS凝胶。为获得更宽的pH范围和凝胶间重现性，还需要进一步研究流速控制和仪器改进.低浓度的MFS凝胶具有较大的孔径，有望提供快速的IEF和HMW蛋白的分离。热聚合可得到1.75%T的聚丙烯酰胺，但太脆，不适合实际应用。琼脂糖的加入在一定程度上对低浓度的MFS凝胶有增强作用。辐射是促进丙烯酰胺聚合的替代方法，其有效地产生自由基。我们利用Co-60 γ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IEF。低浓度MFS凝胶在双向电泳中的应用将研制的低浓度聚丙烯酰胺凝胶用于双向电泳。低浓度的聚丙烯酰胺凝胶在高分子量蛋白质区域的蛋白质斑点更清晰。

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.