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

复丝支撑低浓度聚丙烯酰胺凝胶高性能二维凝胶电泳的开发

• 批准号: 17550072
• 负责人: ODAKE Tamao
• 金额: $ 2.3万
• 依托单位: The University of Tokyo (2006)Gunma University (2005)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17550072/
• 关键词: Electrophoresis; Protein; Proteome; Micro-nano device; Multifilament yarn; Polyacrylamide; Radiation polymerization; Agarose; アクリルアミド; ガンマ線; 蛋白質; 両性担体

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.

我们提出多丝支撑凝胶（MFS凝胶）作为一种新型二维凝胶电泳（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。

项目成果

Development of Easy and Fast Two-Dimensional Electrophoresis Instrument Using Isoelectric Focusing Gel Supported by Yarns

纱线支撑等电聚焦凝胶简易快速二维电泳仪的研制

• 发表时间: 2005
• 期刊: Gunma Pref. Bunseki Kenkyukai Kaiho 31
• 作者: Tamao Odake;Li;Kinichi Tsunoda
• 通讯作者: Kinichi Tsunoda

二次元電気泳動システムにおける等電点電気泳動用ゲル

二维电泳系统中的等电聚焦凝胶

• 发表时间: 2006

MINIATURIZED TWO-DIMENSIONAL GEL ELECTROPHORESIS OF PROTEINS USING LOW-CONCENTRATION MULTIFILAMENT-SUPPORTING GEL FOR ISOELECTRIC FOCUSING

使用低浓度多丝支持凝胶进行等电聚焦的小型化蛋白质二维凝胶电泳

• 发表时间: 2006
• 期刊: Micro Total Analysis Systems 2006 1
• 作者: J.Kawakami;T.Mizuguchi;S.Ito;Tamao Odake
• 通讯作者: Tamao Odake

棒状中空編織品または棒状中空編織品様成形加工品を用いた等電点電気泳動用ゲル

使用棒状中空针织物或棒状中空针织物状成型品的等电聚焦用凝胶

• 发表时间: 2007

MINIATURIZED TWQ-DIMENSIONAL GEL ELECTROPHORESIS OF PROTEINS USING LOW-CONCENTRATION MULTIFILAMENT-SUPPORTING GEL FOR ISOELECTRIC FOCUSING

使用低浓度多丝支持凝胶进行等电聚焦的小型化 TWQ 维凝胶电泳

• 发表时间: 2006
• 期刊: Micro Total Analysis Systems 2006 1
• 作者: Tamao Odake;Kazutoshi Kamezawa;Kozue Miyata;Hiroki Hotta;Yasuaki Kojima;Kin-ichi Tsunoda
• 通讯作者: Kin-ichi Tsunoda