基于免疫印迹定量分析的总蛋白提取方法比较

Comparison of different protein extraction methods using immunoblot quantification analysis

[目的]基于改良的对比蛋白提取结果的方法,探讨两种不同总蛋白提取方法对不同丰度蛋白提取得率的影响。[方法]小胶质细胞株N9总蛋白及商品化预染蛋白标准品,行聚丙烯酰胺凝胶电泳(SDS-PAGE),三种常用图像软件定量免疫印迹结果,比较线性相关系数R^2;选择R^2值最高的软件,灰密度定量不同丰度p-IκBα经RIPA裂解液法和Minute试剂盒法蛋白提取后免疫印迹检测的p-IκBα蛋白得率。[结果]三种软件定量分析同一免疫印迹结果显示,Image Studio Lite的上样量与条带灰密度值的R^2最大(R^2=0.998);运用Image Studio Lite定量免疫印迹检测p-IκBα高丰度组,发现使用RIPA裂解液法比Minute法的p-IκBα提取得率高出7.5%,而p-IκBα低丰度组,前者p-IκBα的得率只有后者的41%,差异显著(P<0.05)。[结论]Minute试剂盒法在免疫印迹检测中对低丰度蛋白具有高提取得率的优越性。

[Objective] Based on the improved method of comparison protein extraction results,to explore the effects of two different total protein extraction methods on the extraction rate of different abundance proteins. [Methods] Whole cell protein extracts from N9 microglial cells and the commercial standard protein were gradiently sampled and separated by 10% SDS-PAGE,followed by three kinds of immunoblot analysis softwares,the software with larger linear correlation coefficients R^2 was employed in the following compare experiment of extraction rates; Based on the suitable immunoblot analysis software,the band intensity of special protein p-IκBα was detected in the samples extracted by RIPA and Minute kit methods. [Results]The results analyzed from Image Studio Lite showed that the gray density value was proportional to the concentration changes of sample loaded,with the greatest linear correlation coefficients（ R^2= 0. 998）; Through Image Studio Lite,in the group with higher level of p-IκBα,the yield of p-IκBα was 7. 5 percent higher in samples extracted by RIPA method than Minute method,In the group with lower level of p-IκBα,the yield of p-IκBα in samples extracted by the RIPA method was just 41 percent of samples extracted by Minute method（ P〈0. 05）. [Conclusion] Compared with RIPA method,Minute method exhibited more advantage for extracting target proteins that were less abundant.