人外周静脉血血清蛋白质双向凝胶电泳条件优化

Optimization of the conditions for serum protein two-dimensional gel electrophoresis in peripheral venous blood of human being

目的优化人外周静脉血血清蛋白质的双向凝胶电泳条件。方法血清总蛋白经去除白蛋白、IgG和丙酮沉淀去盐分处理后,固相pH梯度等电聚焦(IEF)和十二烷基硫酸钠-聚丙烯酰胺凝胶(SDS-PAGE)进行蛋白质电泳,对蛋白质的上样量、固相pH梯度(IPG)胶条的pH值范围、凝胶浓度、水化上样液体积和IEF程序及其参数等条件进行优化。结果将50～100μg蛋白质溶解于终体积为350μl水化上样液,应用pH4～7的IPG胶条和浓度12%的SDS-PAGE凝胶进行双向电泳,经优化电泳电流、时间等参数后,建立了合适的双向凝胶电泳条件,得到比较理想的蛋白质二维图谱。结论优化了血清蛋白质双向凝胶电泳条件,为进一步开展血清差异蛋白质组学研究奠定基础。

Objective To optimize the conditions for serum protein two-dimensional gel electrophoresis（2-DE） in peripherally venous blood of human being. Methods After albumin, IgG and salt were removed from serum samples, immobilized pH gradient isoelectrie focusing（IEF） and vertical sodium dodeeyl sulfonate polyaerylamide gel electrophoresis （SDS-PAGE） were applied to separate proteins. The amount of protein sample, pH range of IPG, the concentration of gel, the volume of sample hydration fluid, the IEF process parameters and other conditions were improved. Results With the conditions of dissolving 50 μg to100 μg proteins to form 350 μl sample fluid hydration, adjusting IPG pH from 4 to 7 and using 12% SDS-PAGE gel, as well as optimizing electrophoresis current, time and other parameters, suitable conditions for 2-DE were established and some ideal two dimensional maps of proteins were acquired. Conclusion The optimizated conditions for serum protein 2-DE in human being are the foundation for further study on serum differential proteomics.