油茶雌蕊蛋白双向电泳分离体系的建立

通过比较不同的蛋白提取方法、裂解液成分、离心速率、等电聚焦程序,以及不同浓度的SDS-PAGE凝胶,建立最适于油茶雌蕊总蛋白的双向电泳体系。结果显示:TCA/丙酮+SDS/酚抽法提取蛋白,裂解液7 mol/L尿素,2 mol/L硫脲,80 mmol/L DTT,4%(W/V)CHAPS,2%(V/V),IPG buffer(p H 3-10L)]复溶蛋白干粉,20 000 r/min离心;等电聚焦程序为:100 V 1 h,200 V 2 h,500 V 3 h,1 000 V 2 h,8 000 V 2 h,8 000 V62 000 Vh,500 V 10 h;10%SDS-PAGE凝胶浓度,为适合油茶雌蕊蛋白的双向电泳体系。这些结果为油茶雌蕊蛋白组学的研究奠定了技术基础。

Establishment of Two-dimensional Electrophoresis System for Camellia oleifera Abel. Pistil Proteins

In order to establish a proper two-dimensional electrophoresis(2-DE)system for the pistil proteins of Camellia oleifera Abel., several different protein extraction methods, lysate components, centrifugal rates, isoelectric focusing electrophoresis(IEF)programs, and the concentrations of SDS-PAGE gel were compared. According to the result of 2-DE, the proper 2-DE system for the pistil proteins of C. oleifera consisted of TCA/acetone + SDS/phenol extraction method for protein extraction, lysate7 mol/L carbamide, 2 mol/L sulfocarbamide, 80 mmol/L DTT, 4%(W/V)CHAPS, 2%(V/V)IPG buffer(pH 3-10 L)] for dissolving protein powder, 20 000 rpm for centrifugal, IEF program(100 V 1 h, 200 V 2 h, 500 V 3 h, 1 000 V 2 h, 8 000 V 2 h, 8 000 V 62 000 Vh, 500 V 10 h), and 10% concentration of SDS-PAGE gel. These results provide a technological basic for the proteomic study of Camellia oleifera pistil.