蛋白电泳凝胶放置时间对蛋白丰度的影响分析

目的利用高分辨力的双向电泳技术检测蛋白电泳后凝胶中的蛋白含量随时间推移的损失情况。方法用双向电泳蛋白裂解液提取鼠疫EV菌株(无毒疫苗株)全菌蛋白并定量。取800μg EV蛋白,平行进行两块双向电泳凝胶电泳,电泳结束后一块凝胶立刻固定并染色,另一块凝胶4℃放置8h后固定并染色,使用ImageMaster 2DElite软件比较分析两块凝胶蛋白点数量及蛋白丰度。结果相比直接固定染色的凝胶,8h后固定染色的凝胶中蛋白点扩散丢失严重,比电泳后直接固定凝胶少346个蛋白点,且部分相邻蛋白点发生融合,含量在20~350ng的低丰度蛋白65.9%因蛋白扩散丢失。结论蛋白凝胶放置8h后大量低丰度蛋白由于扩散会丢失,并直接影响后续免疫印迹、质谱鉴定等实验结果的可靠性。

Analysis of protein abundance with respect to how long electrophoresis gels are left to stand

Objective To use two-dimensional electrophoresis （2DE） to analyze the loss of proteins in electrophoresis gels. Methods Total protein of a vaccine strain of Yersinia pestis （EV strain） was extracted and quantified in parallel with commercial protein extraction and clean-up kits. One 2-DE gel （gel A） was immobilized and stained immediately after electrophoresis while another 2-DE gel （gel B） was immobilized and stained 8 hours after electrophoresis. The results were compared using ImageMaster 2D Elite software. Results Protein loss in gel B was severe. Compared to gel A, gel B had 346 fewer protein spots, and some part of the adjacent spots had merged. Furthermore, about 65.9% of low-abundance proteins were lost on gel B. Conclusion An electrophoresis gel left to stand for more than 8 hours without immo- bilized will have extensive loss of low-abundance proteins, thus affecting the reliability of an experiment＇s results.